Therapy of small-cell lung cancer (sclc) with a topoisomerase-i inhibiting antibody-drug conjugate (adc) targeting trop-2

ABSTRACT

The present invention relates to treatment of SCLC with therapeutic ADCs comprising a drug attached to an anti-Trop-2 antibody or antigen-binding antibody fragment. Preferably, the drug is SN-38. More preferably, the antibody is an hRS7 antibody and the ADC is sacituzumab govitecan. The ADC may be administered at a dosage of between 4 mg/kg and 16 mg/kg, preferably 4, 6, 8, 9, 10, 12, or 16 mg/kg, mostly preferably 8 to 10 mg/kg. When administered at specified dosages and schedules, the ADC can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy. Surprisingly, the ADC is effective to treat cancers that are refractory to or relapsed from irinotecan or topotecan. Preferably, the ADC is administered as a combination therapy with one or more other anti-cancer treatments, such as carboplatin or cisplatinum.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/820,708, filed Nov. 22, 2017, which was acontinuation-in-part of U.S. patent application Ser. No. 15/069,208,filed Mar. 14, 2016, which was a continuation-in-part of U.S. patentapplication Ser. No. 14/667,982 (now issued U.S. Pat. No. 9,493,573),filed Mar. 25, 2015, which was a divisional of U.S. application Ser. No.13/948,732 (now U.S. Pat. No. 9,028,833), filed Jul. 23, 2013, whichclaimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplications 61/736,684, filed Dec. 13, 2012, and 61/749,548, filed Jan.7, 2013. Application Ser. No. 15/069,208 claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Applications 62/133,654, filedMar. 16, 2015, 62/133,729, filed Mar. 16, 2015, 62/138,092, filed Mar.25, 2015, 62/156,608, filed May 4, 2015, and 62/241,881, filed Oct. 15,2015. Application Ser. No. 15/069,208 claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application 62/428,655, filedDec. 1, 2016. The present application claims the benefit under 35 U.S.C.119(e) of U.S. Provisional Patent Application 62/463,316, filed Feb. 24,2017. The text of each priority application is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 14, 2018, isnamed IMM370US1_SL.txt and is 8,305 bytes in size.

FIELD OF THE INVENTION

This invention relates to compositions and methods of use of anti-Trop-2antibody-drug conjugates (ADCs), for treating small cell lung cancer(SCLC). Preferably, the ADC is an anti-Trop-2-SN-38 conjugate, such assacituzumab govitecan. More preferably, a linker such as CL2A may beused to attach the drug to the antibody or antibody fragment. However,other linkers, other known cytotoxic drugs, and other known methods ofconjugating drugs to antibodies may be utilized. Most preferably, theantibody or antigen-binding fragment thereof is a humanized RS7antibody. The antibody or fragment may be attached to 1-12, 1-6, 1-5,6-8 or 7-8 copies of drug moiety or drug-linker moiety per antibody orfragment. The ADCs are of use for therapy of SCLC patients who areeither chemosensitive to or chemoresistant to first-lineplatinum-containing chemotherapy. In certain embodiments, the ADCs maybe of use for first-line therapy of SCLC. Surprisingly, the ADCs are ofuse in SCLC patients who either relapsed from or fail to respond totopetecan therapy, despite it also being an inhibitor of topoisomeraseI. The ADCs are similarly active in second line vs. later stages ofmetastatic SCLC (mSCLC). The ADCs may be used alone or as a combinationtherapy, along with one or more therapeutic modalities selected from thegroup consisting of surgery, radiation therapy, chemotherapy,immunomodulators, cytokines, chemotherapeutic agents, pro-apoptoticagents, anti-angiogenic agents, cytotoxic agents, drugs, toxins,radionuclides, RNAi, siRNA, a second antibody or antibody fragment, andan immunoconjugate. In preferred embodiments, the combination of ADC andother therapeutic modality exhibits a synergistic effect and is moreeffective to induce cancer cell death than either ADC or othertherapeutic modality alone, or the sum of the effects of ADC and othertherapeutic modality administered individually. Surprisingly,subcutaneous administration of the anti-Trop-2 ADC does not result inunacceptable localized toxicity at the site of administration and inalternative embodiments the ADC may be administered intravenously orsubcutaneously.

RELATED ART

Small-cell lung cancer (SCLC), originating from neuroendocrineprogenitor cells, comprises approximately 15% of all lung cancers, yethas one of the lowest 5-year survival rates at 6% (Alvarado-Luna et al.,2016, Transl Lung Cancer Res 5:26-38; Siegel et al., 2017, CA Cancer JClin 67:7-30). This is because it is highly aggressive, with abouttwo-thirds of patients having metastatic disease at diagnosis (Fruh etal., 2013, Ann Oncol 24(6):vi99-105). Whereas first-line therapy ofstage IV SCLC is palliative yet has a high initial response rate of60-75%, the outcome usually is poor, with a median progression-freesurvival (PFS) of only 5.5 months and a median overall survival (OS) of<10 months (Foster et al., 2011, Cancer 117:1262-71; Wolfson et al.,2011, Int J Radiat Oncol Biol Phys 81:77-84) with platinum-basedchemotherapy (Fruh et al., 2013, Ann Oncol 24(6):vi99-105).

Responses to second-line therapy have been poorer, such as <10%, andwith a median survival of only 4 or 5 months after second- or third-linechemotherapy (Hurwitz et al., 2009, Oncologist 14:986-94; Schneider,2008, J Natl Compr Canc Netw 6:323-31), especially when there isresistance to first-line treatment (i.e., response duration <3 months).The only approved drug in this setting, since 1998, is topotecan,indicated for recurrent patients who were sensitive (duration ofresponse exceeding 3 months) to a platinum-based therapy (O'Brien etal., 2006, J Clin Oncol 24:5441-7; Perez-Soler et al., 1996, J ClinOncol 14:2785-90). However, irinotecan, taxanes, vinorelbine, andgemcitabine also are given frequently to patients with chemosensitiverecurrent disease (Furuse et al., 1996, Oncology 53:169-72; Smit et al.,1998, Br J Cancer 77:347-51; Sandler, 2001, Oncology (Williston Park)15:11-2; van der Lee et al., 2001, Ann Oncol 12:557-61).

A review of recent randomized phase II and III clinical trials includingtopotecan in a control arm showed 13 to 17% responses in second line(Inoue et al., 2008, J Clin Oncol 26:5401-6; Jotte et al., 2011, J ClinOncol 29:287-93; Horita et al., 2015, Sci Rep 5:15437), but with as muchas a 20% response rate among patients with chemosensitive disease, andonly 4% for those who were chemoresistant (von Pawel, 2003, Lung Cancer41 (Suppl 4):S3-8). However, these responses and/or diseasestabilization in second line do not translate into improved survival.For example, Hagmann and colleagues (Hagmann et al., 2015, J Cancer6:1148-5418) reported a 22.5% response with topotecan, and a median PFSof 2.4 months and a median OS of 5 months. In a third-line setting, noobjective responses were achieved, while a median PFS of 1.3 months anda median OS of 2.5 months was reported (Hagmann et al., 2015, J Cancer6:1148-5418). Other established single-agent chemotherapies orretreatment with platinum plus etoposide combinations also have beendisappointing, yielding similar survival outcomes as topotecan alone(Hagmann et al., 2015, J Cancer 6:1148-5418). Irinotecan has shown verylow or no responses and a median time to progression (TTP) of 1.7months, while a median OS of 4.6 months also has been reported (Palliset al., 2009, Lung Cancer 65:187-91). Gemcitabine has not given anyobjective response, and resulted in a median TTP of 6 weeks and a medianOS of 6.4 months, while pemetrexed achieved 2 responses among 43patients (ORR, 4%) (Jalal et al., 2009, J Thorac Oncol 4:93-6). Thus,progress in the management of patients with SCLC, especially those withextensive disease, has been disappointing over the past 20 years. Nearlyall patients relapse early and die within a year.

A need exists for more efficacious treatment for patients with SCLC offirst-line or later stages. A particular need exists for bettertherapies for patients who are resistant to standard chemotherapies,such as platinum-containing chemotherapy, topotecan or irinotecan.

SUMMARY

The present invention concerns improved methods and compositions fortreating SCLC, either first-line or else second-line or later. Themethods and compositions are of particular use in treating SCLC patientswho are resistant to standard chemotherapies, such as withplatinum-based or camptothecin compounds like irinotecan and topotecan.The subject methods involve treatment with an anti-Trop-2 ADC,preferably with an anti-Trop-2-SN-38 ADC. In more preferred embodiments,a linker such as a CL2A linker is used to attach the antibody moeity tothe drug moiety. Most preferably, the anti-Trop-2 is a humanized RS7(hRS7) antibody or antigen-binding antibody fragment. The instantmethods and compositions provide substantially improved treatment overthe standard of care for SCLC, with greater efficacy and only manageabletoxicities when used at preferred dosages discussed in detail below.

In various embodiments, the ADC may be used alone or as a combinationtherapy with one or more other therapeutic modalities, such as surgery,radiation therapy, chemotherapy, immunomodulators, cytokines,chemotherapeutic agents, pro-apoptotic agents, anti-angiogenic agents,cytotoxic agents, drugs, toxins, radionuclides, RNAi, siRNA, a secondantibody or antibody fragment, or an immunoconjugate. Preferably, thecombination of ADC and other therapeutic modality is more efficaciousthan either alone, or the sum of the effects of individual treatments.

In a specific embodiment, an anti-Trop-2 antibody may be a humanized RS7antibody (see, e.g., U.S. Pat. No. 7,238,785, the Figures and Examplessection of which are incorporated herein by reference), comprising thelight chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2(SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavychain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG,SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6). However, as discussedbelow other anti-Trop-2 antibodies are known and may be used in thesubject ADCs. A number of cytotoxic drugs of use for cancer treatmentare well-known in the art and any such known drug may be conjugated tothe antibody of interest. In a more preferred embodiment, the drugconjugated to the antibody is a camptothecin, most preferably SN-38(see, e.g., U.S. Pat. No. 9,028,833, the Figures and Examples sectionincorporated herein by reference).

The antibody moiety may be a monoclonal antibody, an antigen-bindingantibody fragment, a bispecific or other multivalent antibody, or otherantibody-based molecule. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. The antibody or fragmentthereof can be a chimeric, a humanized, or a human antibody, as well asvariations thereof, such as half-IgG4 antibodies (referred to as“unibodies”), as described by van der Neut Kolfschoten et al. (Science2007; 317:1554-1557). More preferably, the antibody or fragment thereofmay be designed or selected to comprise human constant region sequencesthat belong to specific allotypes, which may result in reducedimmunogenicity when the ADC is administered to a human subject.Preferred allotypes for administration include a non-G1m1 allotype(nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, theallotype is selected from the group consisting of the nG1m1, G1m3,nG1m1,2 and Km3 allotypes (Jefferies and Lefranc, 2009, mAbs 1(4):1-7).

The drug to be conjugated to the antibody or antibody fragment may beselected from the group consisting of an anthracycline, a camptothecin,a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, anitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, anitrosourea, a triazene, a folic acid analog, a taxane, a COX-2inhibitor, a pyrimidine analog, a purine analog, an antibiotic, anenzyme inhibitor, an epipodophyllotoxin, a platinum coordinationcomplex, a vinca alkaloid, a substituted urea, a methyl hydrazinederivative, an adrenocortical suppressant, a hormone antagonist, anantimetabolite, an alkylating agent, an antimitotic, an anti-angiogenicagent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shockprotein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, apro-apoptotic agent, and a combination thereof.

Specific drugs of use may be selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin,2-pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox(pro-2-PDox), cyano-morpholino doxorubicin, doxorubicin glucuronide,endostatin, epirubicin glucuronide, erlotinib, estramustine,epidophyllotoxin, erlotinib, entinostat, estrogen receptor bindingagents, etoposide (VP16), etoposide glucuronide, etoposide phosphate,exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101,gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,monomethylauristatin F (MMAF), monomethylauristatin D (MMAD),monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib,streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839. Preferably, the drug is SN-38.

Preferred optimal dosing of the subject ADCs may include a dosage ofbetween 4 mg/kg and 18 mg/kg, preferably given either weekly, twiceweekly or every other week. The optimal dosing schedule may includetreatment cycles of two consecutive weeks of therapy followed by one,two, three or four weeks of rest, or alternating weeks of therapy andrest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3 mg/kg,4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. Preferred dosages are4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg. More preferred dosages are 6-12,6-8, 7-8, 8-10, 10-12 or 8-12 mg/kg. The person of ordinary skill willrealize that a variety of factors, such as age, general health, specificorgan function or weight, as well as effects of prior therapy onspecific organ systems (e.g., bone marrow) may be considered inselecting an optimal dosage of ADC, and that the dosage and/or frequencyof administration may be increased or decreased during the course oftherapy. The dosage may be repeated as needed, with evidence of tumorshrinkage observed after as few as 4 to 8 doses. The optimized dosagesand schedules of administration disclosed herein show unexpectedsuperior efficacy and reduced toxicity in human subjects, which couldnot have been predicted from animal model studies. Surprisingly, thesuperior efficacy allows treatment of tumors that were previously foundto be resistant to one or more standard anti-cancer therapies. Moresurprisingly, the treatment has been found effective in tumors that werepreviously resistant to camptothecins, such as irinotecan, the parentcompound of SN-38.

The ADCs are of use for therapy of cancers, such as SCLC and mSCLC. Suchuse may be first-line, second-line, or at later stages of cancerprogression. The compositions and methods of use are efficacious incamptothecin-resistant as well as camptothecin sensitive cancers.Generally, the anti-Trop-2 ADCs are of use for treating any cancer thatexpresses the Trop-2 antigen. However, in a preferred specificembodiment, the cancer is SCLC or mSCLC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with SN-38 conjugates of hRS7(anti-Trop-2), hPAM4 (anti-MUC5ac), hMN-14 (anti-CEACAM5) ornon-specific control hA20 (anti-CD20).

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with anti-TROP2-CL2A-SN-38 conjugatescompared to controls.

FIG. 3A. Structures of CL2-SN-38 and CL2A-SN-38.

FIG. 3B. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using COLO 205 colonicadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. COLO 205 mice (N=6) were treated with 0.4 mg/kgADC and tumors measured twice a week.

FIG. 3C. Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs. CL2Alinkers versus hA20 ADC and saline control, using Capan-1 pancreaticadenocarcinoma. Animals were treated twice weekly for 4 weeks asindicated by the arrows. Capan-1 mice (N=10) were treated with 0.2 mg/kgADC and tumors measured weekly.

FIG. 4A. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Mice bearing Calu-3tumors (N=5-7) were injected with hRS7-CL2-SN-38 every 4 days for atotal of 4 injections (q4dx4).

FIG. 4B. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). COLO 205 tumor-bearingmice (N=5) were injected 8 times (q4dx8) with the ADC or every 2 daysfor a total of 5 injections (q2dx5) with the MTD of irinotecan.

FIG. 4C. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Capan-1 (N=10) weretreated twice weekly for 4 weeks with the agents indicated.

FIG. 4D. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). BxPC-3 tumor-bearingmice (N=10) were treated twice weekly for 4 weeks with the agentsindicated.

FIG. 4E. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). In addition to ADCgiven twice weekly for 4 week, SK-MES-1 tumor-bearing (N=8) micereceived the MTD of CPT-11 (q2dx5).

FIG. 5A. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of AST.

FIG. 5B. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of ALT.

FIG. 5C. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in neutrophil counts in Cynomolgusmonkeys.

FIG. 5D. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in platelet counts in Cynomolgusmonkeys.

FIG. 6. In vitro efficacy of anti-Trop-2-paclitaxel ADC againstMDA-MB-468 human breast adenocarcinoma.

FIG. 7. In vitro efficacy of anti-Trop-2-paclitaxel ADC against BxPC-3human pancreatic adenocarcinoma.

FIG. 8A. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in Capan-1 human pancreatic adenocarcinoma.

FIG. 8B. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in BxPC-3 human pancreatic adenocarcinoma.

FIG. 8C. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in NCI-N87 human gastric adenocarcinoma.

FIG. 9A. Comparison of cytotoxicity of naked or SN-38 conjugated hRS7vs. 162-46.2 antibodies in BxPC-3 human pancreatic adenocarcinoma.

FIG. 9B. Comparison of cytotoxicity of naked or SN-38 conjugated hRS7vs. 162-46.2 antibodies in MDA-MB-468 human breast adenocarcinoma.

FIG. 10. IMMU-132 phase I/II data for best response by RECIST criteria.

FIG. 11. IMMU-132 phase I/II data for time to progression and bestresponse (RECIST).

FIG. 12. Combination therapy with IMMU-132 and carboplatin or cisplatin,compared to IMMU-132, carboplatin or cisplatin alone, a non-targetingADC or saline control.

FIG. 13. Combination therapy with IMMU-132 plus carboplatin, compared toIMMU-132 or caboplatin alone or saline control.

FIG. 14. Combination therapy with IMMU-132 plus cisplatinum, compared toIMMU-132 or cisplatinum alone or saline control.

FIG. 15. Cachexia in mice treated with combination therapy with IMMU-132and carboplatin or cisplatin, compared to IMMU-132, carboplatin orcisplatin alone, or saline control.

FIG. 16. Graphic representation of anti-tumor response and duration inresponse-assessable patients. (A) Best percentage change in the sum ofthe diameters for the selected target lesion and best overall responsedescriptor according to RECIST 1.1 criteria. Patients are identifiedwith respect to the sacituzumab govitecan starting dose and whether theywere sensitive or resistant to prior first-line therapy. Patient withunconfirmed partial responses failed to maintain ≥30% on their next CTassessment 4-6 weeks after the first observed objective response. Thebest overall response for these patients by RECIST 1.0 is stabledisease. (B) Duration of response from the start of treatment for thosepatients who achieved stable disease or better. Timing when tumorshrinkage achieved ≥30% is shown, along with sacituzumab govitecanstarting dose and sensitivity to first-line therapy. (C) Dynamics ofresponse for patients who achieved stable disease or better. Twopatients with confirmed partial responses who are continuing treatmentare shown with dashed line.

FIG. 17. (A) Kaplan-Meier derived progression-free and (B) overallsurvival curves for all 53 SCLC patients enrolled in the sacituzumabgovitecan trial.

FIG. 18. This 64-year-old male diagnosed with advanced SCLC receivedcarboplatin as 1^(st) line therapy from July 2013 to November 2013, withetoposide added in November and December 2013. The disease relapsed inMay 2014. Prior to starting sacituzumab govitecan, the tumor lesions atbaseline (May 2014) included subcarinal lymph node (20 mm) and rightadrenal gland tumors (A) (43×34 mm diameter adrenal mass), as well asmultiple unmeasurable lesions in the right and left lobes of the liver,thickening of the right hilum, a left upper lobe pulmonary nodule, andesophageal thickening. The response evaluation after 2 months of therapyshowed 50% reduction according to RECIST 1.1 (B) (adrenal mass shrinksto 14 mm, subcarinal lymph node shrinks to 17 mm). By the secondresponse assessment, the adrenal mass was no longer visible, while thesubcarinal node experienced its maximum shrinkage ˜11 months from thestart of treatment (to 11 mm), yielding maximum shrinkage of 82%. Thepatient experienced a 21-month duration of response (July 2014 to March2016). (C) Immunohistology of a biopsy from the adrenal mass stained forTrop-2 and scored as 2+, but with sparse distribution among the tumorcells.

FIG. 19. The Figure shows the effects of IMMU-132 therapy in a57-year-old male, 1 pack/day smoker since aged 13, who was diagnosedwith metastatic small cell neuroendocrine carcinoma in 2012, receivedcarboplatin and etoposide as 1^(st) line therapy from October 2012 toJanuary 2013, and upon recurrence, received topotecan from September2013 to November 2013 with no response. Due to progressing disease, thepatient transitioned to a combination of carboplatin and etoposide fromNovember 2013 to April 2014 and further transitioned to paclitaxel fromMay 2014 to June 2014. In September 2014, he commenced on sacituzumabgovitecan achieving a durable response. CT images of 4 of the 5 targetlesions from baseline and after 2 months of treatment, when the sumreduced from 230 mm to 138 mm (40% shrinkage), are shown. (A) Axialslice of the right suprarenal mass measuring 79×65 mm at baseline; (B)showing a reduction to 48×26 mm at first assessment. (C) Left adrenalmass measuring 52×38 mm at baseline; (D) 36×17 mm at first assessment.(E) Left axillary mass at baseline shown in coronal plane (60×55 mm) andafter 2 months of treatment (F), it shrinks to 30×24 mm. (G) Right upperlobe lung mass shown in coronal plane (21×12 mm) shrinking to 16×12 mm(H). (I) Immunohistology of a lung biopsy stained for Trop-2 with arange of staining of tumor cells from negative to 3+, but overall scoredas 2+.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Antibody fragments may also include singledomain antibodies and IgG4 half-molecules, as discussed below.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. The term“antibody fragment” also includes isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”).

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

A bispecific antibody is an antibody that can bind simultaneously to twodifferent targets. Bispecific antibodies (bsAb) and bispecific antibodyfragments (bsFab) may have at least one arm that specifically binds to,for example, a tumor-associated antigen and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

Anti-Trop-2 Antibodies

The subject ADCs may include an antibody or fragment thereof that bindsto Trop-2. In a specific preferred embodiment, the anti-Trop-2 antibodymay be a humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785,incorporated herein by reference in its entirety), comprising the lightchain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful forimmunotoxin production. (Id.) It is well documented that internalizationof immunotoxin conjugates is a requirement for anti-tumor activity.(Pastan et al., Cell 47:641, 1986) Internalization of drugimmunoconjugates has been described as a major factor in anti-tumorefficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988) Thus,the RS7 antibody exhibits several important properties for therapeuticapplications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. U.S. Pat. No. 8,715,662discloses anti-Trop-2 antibodies produced by hybridomas deposited at theAID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD08021. U.S. Patent Application Publ. No. 20120237518 disclosesanti-Trop-2 antibodies 77220, KM4097 and KM4590. U.S. Pat. No. 8,309,094(Wyeth) discloses antibodies A1 and A3, identified by sequence listing.The Examples section of each patent or patent application cited above inthis paragraph is incorporated herein by reference. Non-patentpublication Lipinski et al. (1981, Proc Natl. Acad Sci USA, 78:5147-50)disclosed anti-Trop-2 antibodies 162-25.3 and 162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies in thesubject ADCs.

Use of anti-Trop-2 antibodies has been disclosed for immunotherapeuticsother than ADCs. The murine IgG2a antibody edrecolomab (PANOREX®) hasbeen used for treatment of colorectal cancer, although the murineantibody is not well suited for human clinical use (Baeuerle & Gires,2007, Br. J Cancer 96:417-423). Low-dose subcutaneous administration ofecrecolomab was reported to induce humoral immune responses against thevaccine antigen (Baeuerle & Gires, 2007). Adecatumumab (MT201), a fullyhuman anti-Trop-2 antibody, has been used in metastatic breast cancerand early-stage prostate cancer and is reported to act through ADCC andCDC activity (Baeuerle & Gires, 2007). MT110, a single-chainanti-Trop-2/anti-CD3 bispecific antibody construct has reported efficacyagainst ovarian cancer (Baeuerle & Gires, 2007). Catumaxomab, a hybridmouse/rat antibody with binding affinity for Trop-2, CD3 and Fcreceptor, was reported to be active against ovarian cancer (Baeuerle &Gires, 2007). Proxinium, an immunotoxin comprising anti-Trop-2single-chain antibody fused to Pseudomonas exotoxin, has been tested inhead-and-neck and bladder cancer (Baeuerle & Gires, 2007). None of thesestudies contained any disclosure of the use of anti-Trop-2 antibody-drugconjugates.

Camptothecin Conjugates

Non-limiting methods and compositions for preparing immunoconjugatescomprising a camptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units, mostpreferably containing 6-8 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide(oracetylene) moiety of said defined bifunctional PEG is optionallyattached to the N-terminus of an L-amino acid or a polypeptide, with theC-terminus attached to the amino group of amino alcohol, and the hydroxygroup of the latter is attached to the hydroxyl group of the drug in theform of carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these are deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group is readilydeprotected under physiological pH conditions after the bioconjugate isadministered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. Click chemistry takes place inaqueous solution at near-neutral pH conditions, and is thus amenable fordrug conjugation. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula (1) shown below.

MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (1)

where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 1, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the conjugate of the preferred embodiment of formula 1,m is 0, A′ is L-valinol, and the drug is exemplified by SN-38. Inanother example of formula 1, m is 1 and represented by a derivatizedL-lysine, A′ is L-valinol, and the drug is exemplified by SN-38. In thisembodiment, an amide bond is first formed between the carboxylic acid ofan amino acid such as lysine and the amino group of valinol, usingorthogonal protecting groups for the lysine amino groups. The protectinggroup on the N-terminus of lysine is removed, keeping the protectinggroup on the side chain of lysine intact, and the N-terminus is coupledto the carboxyl group on the defined PEG with azide (or acetylene) atthe other end. The hydroxyl group of valinol is then attached to the20-chloroformate derivative of 10-hydroxy-protected SN-38, and thisintermediate is coupled to an L2 component carrying the antibody-bindingmoiety as well as the complementary acetylene (or azide) group involvedin the click cycloaddition chemistry. Finally, removal of protectinggroups at both lysine side chain and SN-38 gives the product of thisexample.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-3 8 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 1 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody-binding group.

In another example of a preferred embodiment, the A-OH of A′ of generalformula 1 is derived from a substituted 4-aminobenzyl alcohol, and ‘AA’is comprised of a single L-amino acid with m=1 in the general formula 1,and the drug is exemplified with SN-38. Single amino acid of AA may beselected from any one of the following L-amino acids: alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine. Thesubstituent R on 4-aminobenzyl alcohol moiety (A-OH embodiment of A′) ishydrogen or an alkyl group selected from C1-C10 alkyl groups. An exampleof this formula, wherein the single amino acid AA is L-lysine and R═H,and the drug is exemplified by SN-38 is referred to as MAb-CL2A-SN-38(shown below). The structure differs from the linker MAb-CL2-SN-3 8 inthe substitution of a single lysine residue for a Phe-Lys dipeptidefound in the CL2 linker. The Phe-Lys dipeptide was designed as acathepsin B cleavage site for lysosomal enzyme, which was considered tobe important for intracellular release of bound drug. Surprisingly,despite the elimination of the cathepsin-cleavage site, immunoconjugatescomprising a CL2A linker are apparently more efficacious in vivo thanthose comprising a CL2 linker.

In a preferred embodiment, AA comprises a polypeptide moiety, preferablya di, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO: 9)(Trouet et al., 1982).

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 repeating monomeric units. The introduction of PEG may involveusing heterobifunctionalized PEG derivatives which are availablecommercially. The heterobifunctional PEG may contain an azide oracetylene group.

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single antibody-binding moiety. In a representative example, the‘L2’ component is appended to 2 acetylenic groups, resulting in theattachment of two azide-appended SN-38 molecules. The bonding to MAb mayinvolve a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. In a morepreferred embodiment, attachment of SN-38 to reduced disulfidesulfhydryl groups results in formation of an antibody-SN-38immunoconjugate with 6 to 8 SN-38 moieties covalently attached perantibody molecule. Other methods of providing cysteine residues forattachment of drugs or other therapeutic agents are known, such as theuse of cysteine engineered antibodies (see U.S. Pat. No. 7,521,541, theExamples section of which is incorporated herein by reference.)

In alternative preferred embodiments, the chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), Pro-2PDOX, CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin,ansamycins, and epothilones. In a more preferred embodiment, thechemotherapeutic moiety is SN-38. Preferably, in the conjugates of thepreferred embodiments, the antibody links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; most preferably about 6 to about 8 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L2’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of this work, a process was surprisingly discovered bywhich CPT drug-linkers can be prepared wherein CPT additionally has a10-hydroxyl group. This process involves, but is not limited to, theprotection of the 10-hydroxyl group as a t-butyloxycarbonyl (BOC)derivative, followed by the preparation of the penultimate intermediateof the drug-linker conjugate. Usually, removal of the BOC group requirestreatment with strong acid such as trifluoroacetic acid (TFA). Underthese conditions, the CPT 20-O-linker carbonate, containing protectinggroups to be removed, is also susceptible to cleavage, thereby givingrise to unmodified CPT. In fact, the rationale for using a mildlyremovable methoxytrityl (MMT) protecting group for the lysine side chainof the linker molecule, as enunciated in the art, was precisely to avoidthis possibility (Walker et al., 2002). It was discovered that selectiveremoval of phenolic BOC protecting group is possible by carrying outreactions for short durations, optimally 3-to-5 minutes. Under theseconditions, the predominant product was that in which the ‘BOC’ at10-hydroxyl position was removed, while the carbonate at ‘20’ positionwas intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the the final productis ready for conjugation to antibodies without a need for deprotectingthe 10-OH protecting group. The 10-hydroxy protecting group, whichconverts the 10-OH into a phenolic carbonate or a phenolic ester, isreadily deprotected by physiological pH conditions or by esterases afterin vivo administration of the conjugate. The faster removal of aphenolic carbonate at the 10 position vs. a tertiary carbonate at the 20position of 10-hydroxycamptothecin under physiological condition hasbeen described by He et al. (He et al., Bioorganic & Medicinal Chemistry12: 4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be‘COR’ where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—”where n is 1-10 and wherein the terminal amino group is optionally inthe form of a quaternary salt for enhanced aqueous solubility, or asimple alkyl residue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it canbe an alkoxy moiety such as “CH₃—(CH₂)n-O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CHCH₂—CH₂—O—” where R₁ is ethyl or methyl and n isan integer with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 1, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody (indicated herein as “MAb”).

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 1, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the antibody is a monoclonal antibody (MAb). In otherembodiments, the antibody may be a multivalent and/or multispecific MAb.The antibody may be a murine, chimeric, humanized, or human monoclonalantibody, and said antibody may be in intact, fragment (Fab, Fab′,F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form, or ofan IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.

Antibody Preparation

Techniques for preparing monoclonal antibodies against virtually anytarget antigen, such as Trop-2, are well known in the art. See, forexample, Köhler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991). Briefly, monoclonal antibodies can be obtained byinjecting mice with a composition comprising an antigen, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The V_(κ) sequence for the MAb may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the Vκ and V_(H)expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman K and IgG₁ constant region domains.

Humanized Antibodies

The subject ADCs may include an antibody or fragment thereof that bindsto Trop-2. In a specific preferred embodiment, the anti-Trop-2 antibodymay be a humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785,incorporated herein by reference in its entirety), comprising the lightchain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXENOMOUSE® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies and Target Antigens

As discussed above, in preferred embodiments the ADCs are of use fortreatment of cancer. In certain embodiments, the target cancer mayexpress one or more target tumor-associated antigens (TAAs). Particularantibodies that may be of use for therapy of cancer include, but are notlimited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20,anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20),lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor),ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1,also known as Trop-2)), PAM4 or KC4 (both anti-mucin), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5),MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19),TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specificmembrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA),G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin) andtrastuzumab (anti-ErbB2). Preferably, the antibody is an hRS7 antibody.

Such anti-TAA antibodies are known in the art (e.g., U.S. Pat. Nos.5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publ. No.20050271671; 20060193865; 20060210475; 20070087001; the Examples sectionof each incorporated herein by reference.) Specific known antibodies ofuse include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.7,151,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No.7,300,655), hLL1 (U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No.5,789,554), hMu-9 (U.S. Pat. No. 7,387,772), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.8,287,865), hR1 (U.S. Pat. No. 9,441,043), hRS7 (U.S. Pat. No.7,238,785), hMN-3 (U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406) and D2/B (WO 2009/130575) the text of each recited patent orapplication is incorporated herein by reference with respect to theFigures and Examples sections.

Other useful tumor-associated antigens that may be targeted includecarbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1,CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20(e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55,CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133,CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP),VEGF (e.g., AVASTIN®, fibronectin splice variant), ED-B fibronectin(e.g., L19), EGP-1 (Trop-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1)(e.g., ERBITUX®), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor,Ga 733, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24,HER-2/neu, histone H2B, histone H3, histone H4, insulin-like growthfactor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigento which L243 binds, CD66 antigens, i.e., CD66a-d or a combinationthereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophagemigration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac,placental growth factor (PlGF), PSA (prostate-specific antigen), PSMA,PAM4 antigen, PD-1 receptor, PD-L1, NCA-95, NCA-90, A3, A33, Ep-CAM,KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

Cancer stem cells, which are ascribed to be more therapy-resistantprecursor malignant cell populations (Hill and Perris, J. Natl. CancerInst. 2007; 99:1435-40), have antigens that can be targeted in certaincancer types, such as CD133 in prostate cancer (Maitland et al., ErnstSchering Found. Sympos. Proc. 2006; 5:155-79), non-small-cell lungcancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91), andglioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 incolorectal cancer (Dalerba et al., Proc. Natl. Acad. Sci. USA 2007;104(24)10158-63), pancreatic cancer (Li et al., Cancer Res. 2007;67(3):1030-7), and in head and neck squamous cell carcinoma (Prince etal., Proc. Natl. Acad. Sci. USA 2007; 104(3)973-8). Another usefultarget for breast cancer therapy is the LIV-1 antigen described byTaylor et al. (Biochem. J. 2003; 375:51-9).

Checkpoint inhibitor antibodies have been used in cancer therapy. Immunecheckpoints refer to inhibitory pathways in the immune system that areresponsible for maintaining self-tolerance and modulating the degree ofimmune system response to minimize peripheral tissue damage. However,tumor cells can also activate immune system checkpoints to decrease theeffectiveness of immune response against tumor tissues. Exemplarycheckpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4(CTLA4, also known as CD152), programmed cell death protein 1 (PD1, alsoknown as CD279) and programmed cell death 1 ligand 1 (PD-L1, also knownas CD274), may be used in combination with one or more other agents toenhance the effectiveness of immune response against disease cells,tissues or pathogens. Exemplary anti-PD1 antibodies includelambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERSSQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.).Anti-PD1 antibodies are commercially available, for example from ABCAM®(AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE(J105, J116, MIH4). Exemplary anti-PD-L1 antibodies include MDX-1105(MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559(BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodies are also commerciallyavailable, for example from AFFYMETRIX EBIOSCIENCE (MIH1). Exemplaryanti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) andtremelimumab (PFIZER). Anti-PD1 antibodies are commercially available,for example from ABCAM® (AB134090), SINO BIOLOGICAL INC. (11159-H03H,11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967,PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recently received FDAapproval for treatment of metastatic melanoma (Wada et al., 2013, JTransl Med 11:89).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, and colon (e.g., Meyer-Siegler et al.,2004, BMC Cancer 12:34; Shachar & Haran, 2011, LeukLymphoma 52:1446-54).Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of therapeutic usefor treatment of MIF-mediated diseases.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.)

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including tumor-associated antigens, have been deposited at theATCC and/or have published variable region sequences and are availablefor use in the claimed methods and compositions. See, e.g., U.S. Pat.Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206′ 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338. These are exemplary only anda wide variety of other antibodies and their hybridomas are known in theart. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown belowfor the exemplary antibodies rituximab (SEQ ID NO:7) and veltuzumab (SEQID NO:8).

Rituximab heavy chain variable region sequence (SEQ ID NO: 7)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CHI) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes 356/358 431 Complete allotype 214 (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Nanobodies

Nanobodies are single-domain antibodies of about 12-15 kDa in size(about 110 amino acids in length). Nanobodies can selectively bind totarget antigens, like full-size antibodies, and have similar affinitiesfor antigens. However, because of their much smaller size, they may becapable of better penetration into solid tumors. The smaller size alsocontributes to the stability of the nanobody, which is more resistant topH and temperature extremes than full size antibodies (Van Der Linden etal., 1999, Biochim Biophys Act 1431:37-46). Single-domain antibodieswere originally developed following the discovery that camelids (camels,alpacas, llamas) possess fully functional antibodies without lightchains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77:13-22).The heavy-chain antibodies consist of a single variable domain (V_(HH))and two constant domains (C_(H)2 and C_(H)3). Like antibodies,nanobodies may be developed and used as multivalent and/or bispecificconstructs. Humanized forms of nanobodies are in commercial developmentthat are targeted to a variety of target antigens, such as IL-6R, vWF,TNF, RSV, RANKL, IL-17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium),with potential clinical use in cancer and other disorders (e.g., Saerenset al., 2008, Curr Opin Pharmacol 8:600-8; Muyldermans, 2013, Ann RevBiochem 82:775-97; Ibanez et al., 2011, J Infect Dis 203:1063-72).

The plasma half-life of nanobodies is shorter than that of full-sizeantibodies, with elimination primarily by the renal route. Because theylack an Fc region, they do not exhibit complement dependentcytotoxicity.

Nanobodies may be produced by immunization of camels, llamas, alpacas orsharks with target antigen, following by isolation of mRNA, cloning intolibraries and screening for antigen binding. Nanobody sequences may behumanized by standard techniques (e.g., Jones et al., 1986, Nature 321:522, Riechmann et al., 1988, Nature 332: 323, Verhoeyen et al., 1988,Science 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89:4285, Sandhu, 1992, Crit. Rev. Biotech. 12: 437, Singer et al., 1993, J.Immun. 150: 2844). Humanization is relatively straight-forward becauseof the high homology between camelid and human FR sequences.

In various embodiments, the subject ADCs may comprise nanobodies fortargeted delivery of conjugated drug to targeted cancer cells.Nanobodies of use are disclosed, for example, in U.S. Pat. Nos.7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398; 8,557,965;8,623,361 and 8,629,244, the Examples section of each incorporatedherein by reference.)

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 kDfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3×anti-Trop-2antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against a B-cellassociated antigen, such as anti-CD3×anti-CD19, anti-CD3×anti-CD20,anti-CD3×anti-CD22, anti-CD3×anti-HLA-DR or anti-CD3×anti-CD74. Incertain embodiments, the techniques and compositions for therapeuticagent conjugation disclosed herein may be used with bispecific ormultispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabody) andtetramers (termed tetrabody) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

Conjugation Protocols

Antibodies or fragments thereof may be conjugated to one or moretherapeutic or diagnostic agents. The therapeutic agents do not need tobe the same but can be different, e.g. a drug and a radioisotope. Forexample, ¹³¹I can be incorporated into a tyrosine of an antibody orfusion protein and a drug attached to an epsilon amino group of a lysineresidue. Therapeutic and diagnostic agents also can be attached, forexample to reduced SH groups and/or to carbohydrate side chains. Manymethods for making covalent or non-covalent conjugates of therapeutic ordiagnostic agents with antibodies or fusion proteins are known in theart and any such known method may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tomoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Alternative methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering to a subject a therapeuticallyeffective amount of an antibody-drug conjugate (ADC) as describedherein. Diseases that may be treated with the ADCs described hereininclude, but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cellleukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb(epratuzumab, see U.S. Pat. No. 6,183,744), against another CD22 epitope(hRFB4) or antibodies against other B cell antigens, such as CD19, CD20,CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Otherdiseases include, but are not limited to, adenocarcinomas ofendodermally-derived digestive system epithelia, cancers such as breastcancer and non-small cell lung cancer, and other carcinomas, sarcomas,glial tumors, myeloid leukemias, etc. In particular, antibodies againstan antigen, e.g., an oncofetal antigen, produced by or associated with amalignant solid tumor or hematopoietic neoplasm, e.g., agastrointestinal, stomach, colon, esophageal, liver, lung, breast,pancreatic, liver, prostate, ovarian, testicular, brain, bone,urothelial or lymphatic tumor, a sarcoma or a melanoma, areadvantageously used. Such therapeutics can be given once or repeatedly,depending on the disease state and tolerability of the conjugate, andcan also be used optionally in combination with other therapeuticmodalities, such as surgery, external radiation, radioimmunotherapy,immunotherapy, chemotherapy, antisense therapy, interference RNAtherapy, gene therapy, and the like. Each combination will be adapted tothe tumor type, stage, patient condition and prior therapy, and otherfactors considered by the managing physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, therapeutic conjugates comprising ananti-Trop-2 antibody such as the hRS7 MAb can be used to treatcarcinomas such as carcinomas of the esophagus, pancreas, lung, stomach,colon and rectum, urinary bladder, breast, ovary, uterus, kidney andprostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964 and8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3(QQHYITPLT, SEQ ID NO:3) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV,SEQ ID NO:6)

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ¹¹³mIn, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate. Alternatively, one ormore therapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

In preferred embodiments, a therapeutic agent to be used in combinationwith a DNA-breaking antibody conjugate (e.g., an SN-38-ADC) is amicrotubule inhibitor, such as a vinca alkaloid, a taxanes, amaytansinoid or an auristatin. Exemplary known microtubule inhibitorsinclude paclitaxel, vincristine, vinblastine, mertansine, epothilone,docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980,phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010,methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.

In an alternative preferred embodiment, a therapeutic agent to be usedin combination with a DNA-breaking ADC, such as an SN-38-antibodyconjugate, is a PARP inhibitor, such as olaparib, talazoparib (BMN-673),rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699,BSI-201, CEP-8983 or 3-aminobenzamide.

In another alternative, a therapeutic agent used in combination with anantibody or immunoconjugate is a tyrosine kinase inhibitor, such as suchas ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059,GDC-0834, LFM-A13 or RN486.

In yet another alternative, a therapeutic agent used in combination withan antibody or immunoconjugate is a PI3K inhibitor, such as idelalisib,Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib),BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120,XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114,TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, ranpirnase, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. (See, e.g., Pastan. et al., Cell(1986), 47:641, and Sharkey and Goldenberg, CA Cancer J Clin. 2006July-August; 56(4):226-43.) Additional toxins suitable for use hereinare known to those of skill in the art and are disclosed in U.S. Pat.No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and —II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, -γ and -λ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-β,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subjectimmunoconjugates, comprising a camptothecin conjugated to an antibody orantibody fragment, may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibody-drugimmunoconjugates.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

In a preferred embodiment, the immunoconjugate is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of immunoconjugate thatis in the range of from about 1 mg/kg to 24 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may beused. However, in preferred embodiments, the dosage range may be 4 to 16mg/kg, 6 to 12 mg/kg or 8 to 10 mg/kg.

The dosage is preferably administered multiple times, once or twice aweek. A minimum dosage schedule of 4 weeks, more preferably 8 weeks,more preferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 12 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenström's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis. Inpreferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with immunoconjugates mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating cancer in a patient. Exemplary kits may contain at least onedrug-conjugated antibody as described herein. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims.

Example 1. Targeted Therapy of GI Cancers with IMMU-132 (SacituzumabGovitecan), an Anti-Trop-2-SN-38 Antibody Drug Conjugate (ADC)

Trop-2 is a tumor-associated glycoprotein highly prevalent in manyepithelial cancers. Its elevated expression has been linked to moreaggressive disease and a poor prognosis. A humanized mAb binding to theextracellular domain of Trop-2 was conjugated to SN-38 (IMMU-132;average drug:mAb ratio=7.6), the active principle of CPT-11. Afterpotent activity in human tumor xenografts, a Phase I/II trial wasinitiated in patients (pts) with diverse solid tumors, including GIcancers.

Methods:

Patients with metastatic cancers were enrolled after failing standardtherapy, starting at a dose of 8.0 mg/kg given on days 1 and 8 of a3-week cycle. The MTD was determined to be 12 mg/kg; dose levels of 8and 10 mg/kg were chosen for Phase II testing.

Results:

Sixty patients with advanced GI cancers were enrolled in a Phase I/IItrial. In 29 CRC patients (9 treated at 10 mg/kg, 20 at 8 mg/kg), 1 hada PR (partial response) and 14 had SD (stable disease) as the bestresponse by RECIST, with a time to progression (TTP) of 50+ wks for thePR (−65%) and a median of 21+ wks for the SD patients (5 continuing).Thirteen CRC patients had KRAS mutations, 7 showing SD with a median TTPof 19.1+ wks (range, 12.0-34.0; 3 continuing). Of 15 pancreatic cancerpatients that were treated (5 at 8, 7 at 10, and 3 at 12 mg/kg), 7 hadSD as best response for a median TTP of 15.0 wks. Among 11 patients withesophageal cancer (9 started at 8, 1 at 10, and 1 at 18 mg/kg), 8 had CTassessment, showing 1 PR with a TTP of 30+ wks, and 4 with SD of 17.4+,21.9, 26.3, and 29.9 wks. Of 5 gastric cancer patients (2 at 8 and 3 at10 mg/kg), only 3 have had CT assessment, all with SD (1 with 19% targetlesion reduction and an ongoing TTP of 29+ wks).

Neutropenia was the principal dose-limiting toxicity, with fatigue,diarrhea, nausea, and vomiting as other commonly reported toxicities.However, the toxicity profile from 75 patients in the full trial showedonly 17.3% and 2.7% Grade 3 and Grade 4 neutropenia, respectively, andjust 6.7% Grade 3 diarrhea.

Conclusions:

IMMU-132 showed a high therapeutic index in patients with diverserelapsed metastatic GI cancers. It has a moderately-toxic drugconjugated to an internalizing, cancer-selective mAb, which can be givenrepeatedly over many months once weekly×2 in a 21-day cycle.

Example 2. Production and Use of Anti-Trop-2-SN-38 Antibody-DrugConjugate

The humanized RS7 (hRS7) anti-Trop-2 antibody was produced as describedin U.S. Pat. No. 7,238,785, the Figures and Examples section of whichare incorporated herein by reference. SN-38 attached to a CL2A linkerwas produced and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac),hA20 (anti-CD20) or hMN-14 (anti-CEACAM5) antibodies according to U.S.Pat. No. 7,999,083 (Example 10 and 12 of which are incorporated hereinby reference). The conjugation protocol resulted in a ratio of about 6SN-38 molecules attached per antibody molecule.

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4(anti-MUC-5ac), and hMN-14 (anti-CEACAM5) antibodies showed betterefficacies than control hA20-CL2A-SN-38 conjugate (anti-CD20) anduntreated control. Similarly in a BXPC3 model of human pancreaticcancer, the specific hRS7-CL2A-SN-38 showed better therapeutic efficacythan control treatments (FIG. 2).

Example 3. Efficacy of Anti-Trop-2-SN-38 ADC Against Diverse EpithelialCancers In Vivo

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 (hRS7) ADC against several human solid tumor types,and to assess its tolerability in mice and monkeys, the latter withtissue cross-reactivity to hRS7 similar to humans. Two SN-3 8derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The immunoconjugates werecharacterized in vitro for stability, binding, and cytotoxicity.Efficacy was tested in five different human solid tumor-xenograft modelsthat expressed Trop-2 antigen. Toxicity was assessed in mice and inCynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≤0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

In summary, the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant andspecific antitumor effects against a range of human solid tumor types.It was well tolerated in monkeys, with tissue Trop-2 expression similarto humans, at clinically relevant doses.

Introduction

Successful irinotecan treatment of patients with solid tumors has beenlimited, due in large part to the low conversion rate of the CPT-11prodrug into the active SN-3 8 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., <3.2 mg/kg cumulative SN-38 equivalent dose). Trop-2 is widelyexpressed in many epithelial cancers, but also some normal tissues, andtherefore a dose escalation study in Cynomolgus monkeys was performed toassess the clinical safety of this conjugate. Monkeys tolerated 24 mgSN-38 equivalents/kg with only minor, reversible, toxicities. Given itstumor-targeting and safety profile, hRS7-SN-38 provides a significantimprovement in the management of solid tumors responsive to irinotecan.

Material and Methods

Cell Lines, Antibodies, and Chemotherapeutics—

All human cancer cell lines used in this study were purchased from theAmerican Type Culture Collection. These include Calu-3 (non-small celllung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205(colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreaticadenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22(hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc.Irinotecan (20 mg/mL) was obtained from Hospira, Inc.

SN-38 Immunoconjugates and In Vitro Aspects—

Synthesis of CL2-SN-38 has been described previously (Moon et al., 2008,J Med Chem 51:6916-26). Its conjugation to hRS7 IgG and serum stabilitywere performed as described (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Chem Res 15:6052-61). Preparations ofCL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding,and cytotoxicity studies, were conducted as described in the precedingExamples.

In Vivo Therapeutic Studies—

For all animal studies, the doses of SN-38 immunoconjugates andirinotecan are shown in SN-38 equivalents. Based on a mean SN-38/IgGsubstitution ratio of 6, a dose of 500 μg ADC to a 20-g mouse (25 mg/kg)contains 0.4 mg/kg of SN-38. Irinotecan doses are likewise shown asSN-38 equivalents (i.e., 40 mg irinotecan/kg is equivalent to 24 mg/kgof SN-38).

NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, and maleSwiss-Webster mice, 10 weeks old, were purchased from Taconic Farms.Tolerability studies were performed in Cynomolgus monkeys (Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd.

Animals were implanted subcutaneously with different human cancer celllines. Tumor volume (TV) was determined by measurements in 2 dimensionsusing calipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized hRS7-CL2A-SN-38 and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics—

Growth curves are shown as percent change in initial TV over time.Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. An f-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P≤0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and Biodistribution—

¹¹¹In-radiolabeled hRS7-CL2A-SN-38 and hRS7 IgG were injected into nudemice bearing s.c. SK-MES-1 tumors (˜0.3 cm³). One group was injectedintravenously with 20 μCi (250-μg protein) of ¹¹¹In-hRS7-CL2A-SN-38,whereas another group received 20 μCi (250-μg protein) of ¹¹¹In-hRS7IgG. At various timepoints mice (5 per timepoint) were anesthetized,bled via intracardiac puncture, and then euthanized. Tumors and varioustissues were removed, weighed, and counted by γ scintillation todetermine the percentage injected dose per gram tissue (% ID/g). A thirdgroup was injected with 250 μg of unlabeled hRS7-CL2A-SN-38 3 daysbefore the administration of ¹¹¹In-hRS7-CL2A-SN-38 and likewisenecropsied. A 2-tailed t-test was used to compare hRS7-CL2A-SN-38 andhRS7 IgG uptake after determining equality of variance using the f-test.Pharmacokinetic analysis on blood clearance was performed usingWinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys—

Briefly, mice were sorted into 4 groups each to receive 2-mL i.p.injections of either a sodium acetate buffer control or 3 differentdoses of hRS7-CL2A-SN-38 (4, 8, or 12 mg/kg of SN-38) on days 0 and 3followed by blood and serum collection, as described in Results.Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2different doses of hRS7-CL2A-SN-38. Dosages, times, and number ofmonkeys bled for evaluation of possible hematologic toxicities and serumchemistries are described in the Results.

Results

Stability and Potency of hRS7-CL2A-SN-38—

Two different linkages were used to conjugate SN-38 to hRS7 IgG (FIG.3A). The first is termed CL2-SN-38 and has been described previously(Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, ClinChem Res 15:6052-61). A change in the synthesis of CL2 to remove thephenylalanine moiety within the linker was used to produce the CL2Alinker. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity. This result was surprising, since the phenylalanineresidue in CL2 is part of a designed cleavage site for cathepsin B, alysosomal protease.

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 (FIG. 3B) or Capan-1 tumors (FIG. 3C), using 0.4mg or 0.2 mg/kg SN-38 twice weekly×4 weeks, respectively, and withstarting tumors of 0.25 cm³ size in both studies. Both the hRS7-CL2A andCL2-SN-38 conjugates significantly inhibited tumor growth compared tountreated (AUC_(14days)P<0.002 vs. saline in COLO 205 model;AUC_(21days) P<0.001 vs. saline in Capan-1 model), and a nontargetinganti-CD20 control ADC, hA20-CL2A-SN-38 (AUC_(14days) P<0.003 in COLO-205model; AUC_(35days): P<0.002 in Capan-l model). At the end of the study(day 140) in the Capan-1 model, 50% of the mice treated withhRS7-CL2A-SN-38 and 40% of the hRS7-CL2-SN-38 mice were tumor-free,whereas only 20% of the hA20-ADC-treated animals had no visible sign ofdisease. As demonstrated in FIG. 3, the CL2A linker resulted in asomewhat higher efficacy compared to CL2.

Mechanism of Action—

In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC₅₀values in the nmol/L range against several different solid tumor lines(Table 2). The IC₅₀ with free SN-38 was lower than the conjugate in allcell lines. Although there was no apparent correlation between Trop-2expression and sensitivity to hRS7-CL2A-SN-38, the IC₅₀ ratio of the ADCversus free SN-3 8 was lower in the higher Trop-2-expressing cells, mostlikely reflecting the enhanced ability to internalize the drug when moreantigen is present.

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis (e.g., Cusack et al., 2001, Cancer Res 61:3535-40; Liu etal. 2009, Cancer Lett 274:47-53; Lagadec et al., 2008, Br J Cancer98:335-44). Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression (not shown), whereas hRS7-CL2A-SN-38 resultedin only a 10-fold increase (not shown), a finding consistent with thehigher activity with free SN-38 in this cell line (Table 2). However,hRS7-CL2A-SN-38 increased p21^(Waf1/Cip1) expression in Calu-3 more than2-fold over free SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression (not shown). In bothBxPC-3 and Calu-3, upregulation of p53 with free SN-38 was not evidentuntil 48 hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours(not shown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure (not shown). In terms of laterapoptotic events, cleavage of PARP was evident in both cell lines whenincubated with either SN-38 or the conjugate (not shown). The presenceof the cleaved PARP was higher at 24 hours in BxPC-3 (not shown), whichcorrelates with high expression of p21 and its lower IC₅₀. The higherdegree of cleavage with free SN-38 over the ADC was consistent with thecytotoxicity findings.

Efficacy of hRS7-SN-38—

Because Trop-2 is widely expressed in several human carcinomas, studieswere performed in several different human cancer models, which startedusing the hRS7-CL2-SN-38 linkage, but later, conjugates with theCL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mg SN-38/kgof the hRS7-CL2-SN-38 every 4 days×4 had a significantly improvedresponse compared to animals administered the equivalent amount ofnon-targeting hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs. 0.80+0.91 cm³,respectively; AUC_(42days) P<0.026; FIG. 4A). A dose-response wasobserved when the dose was increased to 0.4 mg/kg SN-38 (FIG. 4A). Atthis higher dose level, all mice given the specific hRS7 conjugate were“cured” within 28 days, and remained tumor-free until the end of thestudy on day 147, whereas tumors regrew in animals treated with theirrelevant ADC (specific vs. irrelevant AUC_(98days): P=0.05). In micereceiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5-foldby day 56 (TV=1.10±0.88 cm³; AUC_(56days) P<0.006 vs. hRS7-CL2-SN-38)(FIG. 4A).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG. 4Bhttp://clincancerres.aacrioumals.org/content/17/10/3157.long—F3),hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the 28-daytreatment period with significantly smaller tumors compared to controlanti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³, 1.19±0.59cm³, and 1.77±0.93 cm³, respectively; AUC_(28days) P<0.016).

TABLE 2 Expression of Trop-2 in vitro cytotoxicity of SN-38 andhRS7-SN-38 in various solid tumor lines Cytotoxicity results Trop-2expression hRS7-SN- via FACS SN-38 95% CI 38 95% CI Median fluorescencePercent IC₅₀ IC₅₀ IC₅₀ IC₅₀ ADC/free SN-38 Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) ratio Calu-3 282.2 (4.7)99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95 1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99 5.02-9.72 2.00 PC-3  46.2 (5.5) 73.6% 1.86 1.16-2.99 4.242.99-6.01 2.28 SK-MES-1  44.0 (3.5) 91.2% 8.61  6.30-11.76 23.1417.98-29.78 2.69 BxPC-3  26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.982.80

The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38 in COLO 205 cells, because mouse serum can moreefficiently convert irinotecan to SN-38 (Morton et al., 2000, Cancer Res60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 μgcumulative) was 37.5-fold greater than with the conjugate (64 μg total).

Animals bearing Capan-1 (FIG. 4C) showed no significant response toirinotecan alone when given at an SN-38-dose equivalent to thehRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was0.04±0.05 cm³ in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62cm³ in irinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001; FIG. 4C). When the irinotecan dose was increased 10-fold to 4mg/kg SN-38, the response improved, but still was not as significant asthe conjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49) P<0.001) (FIG. 4C). An equal dose ofnontargeting hA20-CL2-SN-38 also had a significant antitumor effect ascompared to irinotecan-treated animals, but the specific hRS7 conjugatewas significantly better than the irrelevant ADC (TV=0.17+0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49) P<0.018) (FIG. 4C).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 4D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively; AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38 equivalent dose(TV=0.27+0.18 cm³ vs. 0.90+0.62 cm³, respectively; AUC_(day25) P<0.004)(FIG. 4D). Interestingly, in mice bearing SK-MES-1 human squamous celllung tumors treated with 0.4 mg/kg of the ADC (FIG. 4E), tumor growthinhibition was superior to saline or unconjugated hRS7 IgG (TV=0.36±0.25cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively; AUC₂,8_(d),P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD of irinotecanprovided the same antitumor effects as the specific hRS7-SN-38 conjugate(FIG. 5E).

In all murine studies, the hRS7-SN-38 ADC was well tolerated in terms ofbody weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38—

The biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG werecompared in mice bearing SK-MES-1 human squamous cell lung carcinomaxenografts (not shown), using the respective ¹¹¹In-labeled substrates. Apharmacokinetic analysis was performed to determine the clearance ofhRS7-CL2A-SN-38 relative to unconjugated hRS7 (not shown). The ADCcleared faster than the equivalent amount of unconjugated hRS7, with theADC exhibiting ˜40% shorter half-life and mean residence time.Nonetheless, this had a minimal impact on tumor uptake (not shown).Although there were significant differences at the 24- and 48-hourtimepoints, by 72 hours (peak uptake) the amounts of both agents in thetumor were similar. Among the normal tissues, hepatic and splenicdifferences were the most striking (not shown). At 24 hourspostinjection, there was >2-fold more hRS7-CL2A-SN-38 in the liver thanhRS7 IgG (not shown). Conversely, in the spleen there was 3-fold moreparental hRS7 IgG present at peak uptake (48-hour timepoint) thanhRS7-CL2A-SN-38 (not shown). Uptake and clearance in the rest of thetissues generally reflected differences in the blood concentration (notshown).

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123; notshown http://clincancerres.aacrjournals.org/content/17/10/3157.long—F4).Predosing had no appreciable impact on blood clearance or tissue uptake(not shown). These studies suggest that in some tumor models, tumoraccretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transamninase (AST, FIG. 5A) andalanine transaminase (ALT, FIG. 5B). Seven days after treatment, ASTrose above normal levels (>298 U/L) in all 3 treatment groups (FIG. 5A),with the largest proportion of mice being in the 2×8 mg/kg group.However, by 15 days posttreatment, most animals were within the normalrange. ALT levels were also above the normal range (>77 U/L) within 7days of treatment (FIG. 5B) and with evidence of normalization by Day15. Livers from all these mice did not show histologic evidence oftissue damage (not shown). In terms of renal function, only glucose andchloride levels were somewhat elevated in the treated groups. At 2×8mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of273-320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by15 days postinjection. Similarly, chloride levels were slightlyelevated, ranging from 116 to 127 mmol/L (upper end of normal range 115mmol/L) in the 2 highest dosage groups (57% in the 2×8 mg/kg group and100% of the mice in the 2×12 mg/kg group), and remained elevated out to15 days postinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express Trop-2 identified by hRS7, a more suitablemodel was required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (neutrophil and platelet data shown in FIG. 5Cand FIG. 5D), but values did not fall below normal ranges. No abnormalvalues were found in the serum chemistries. Histopathology of theanimals necropsied on day 11 (8 days after last injection) showedmicroscopic changes in hematopoietic organs (thymus, mandibular andmesenteric lymph nodes, spleen, and bone marrow), gastrointestinalorgans (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum),female reproductive organs (ovary, uterus, and vagina), and at theinjection site. These changes ranged from minimal to moderate and werefully reversed at the end of the recovery period (day 32) in alltissues, except in the thymus and gastrointestinal tract, which weretrending towards full recovery at this later timepoint (not shown).

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group (not shown). These dataindicate that dose-limiting toxicities were identical to that ofirinotecan; namely, intestinal and hematologic. Thus, the MTD forhRS7-CL2A-SN-38 lies between 2×0.96 and 1.92 mg SN-38/kg, whichrepresents a human equivalent dose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents (Ohmachi etal., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer99:1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14). TheRS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995,Cancer Res 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38(Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. To simplify the synthetic process, inCL2A the phenylalanine was eliminated, and thus the cathepsin B cleavagesite was removed. Interestingly, this product had a better-definedchromatographic profile compared to the broad profile obtained with CL2(not shown), but more importantly, this change had no impact on theconjugate's binding, stability, or potency in side-by-side testing.These data suggest that SN-38 in CL2 was released from the conjugateprimarily by the cleavage at the pH-sensitive benzyl carbonate bond toSN-38's lactone ring and not the cathepsin B cleavage site.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-3 8 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96, Zhao etal., 2008, Bioconjug Chem 19:849-59) and NK012 (Koizumi et al., 2006,Cancer Res 66:10048-56). ENZ-2208 utilizes a branched PEG to link about3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micellenanoparticle containing 20% SN-38 by weight. With our ADC, thisdisparity (i.e., ratio of potency with free vs. conjugated SN-38)decreased as the Trop-2 expression levels increased in the tumor cells,suggesting an advantage to targeted delivery of the drug. In terms of invitro serum stability, both the CL2- and CL2A-SN-38 forms of hRS7-SN-38yielded a t/_(1/2) of ˜20 hours, which is in contrast to the shortt/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38or CL2A-SN-3 8) significantly inhibited tumor growth in 5 differenttumor models. In 4 of them, tumor regressions were observed, and in thecase of Calu-3, all mice receiving the highest dose of hRS7-SN-38 weretumor-free at the conclusion of study. Unlike in humans, irinotecan isvery efficiently converted to SN-3 8 by a plasma esterase in mice, witha greater than 50% conversion rate, and yielding higher efficacy in micethan in humans (Morton et al., 2000, Cancer Res 60:4206-10; Furman etal., 1999, J Clin Oncol 17:1815-24). When irinotecan was administered at10-fold higher or equivalent SN-38 levels, hRS7-SN-38 was significantlybetter in controlling tumor growth. Only when irinotecan wasadministered at its MTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did itequal the effectiveness of hRS7-SN-38. In patients, we would expect thisadvantage to favor hRS7-CL2A-SN-38 even more, because the bioconversionof irinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues (Jain, 1994, Sci Am 271:58-61). With our conjugate, 50%of the SN-38 will be released in ˜13 hours when the pH is lowered to alevel mimicking lysosomal levels (e.g., pH 5.3 at 37° C.; data notshown), whereas at the neutral pH of serum, the release rate is reducednearly 2-fold. If an irrelevant conjugate enters an acidic tumormicroenvironment, it is expected to release some SN-38 locally. Otherfactors, such as tumor physiology and innate sensitivities to the drug,will also play a role in defining this “baseline” activity. However, aspecific conjugate with a longer residence time should have enhancedpotency over this baseline response as long as there is ample antigen tocapture the specific antibody. Biodistribution studies in the SK-MES-1model also showed that if tumor antigen becomes saturated as aconsequence of successive dosing, tumor uptake of the specific conjugateis reduced, which yields therapeutic results similar to that found withan irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5 (Sapra et al., 2008, Clin Cancer Res 14:1888-96; Pastorini etal., 2010, Clin Cancer Res 16:4809-21), and preclinical studies withNK012 involved its MTD of 30 mg/kg×3 (Koizumi et al., 2006, Cancer Res66:10048-56). Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wasimportant to perform toxicity studies in monkeys that have a similartissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose(˜12 mg/m²) with mild and reversible toxicity, which extrapolates to ahuman dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a Phase I clinical trialof NK012, patients with solid tumors tolerated 28 mg/m² of SN-38 every 3weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchiet al., 2010, Clin Cancer Res 16:5058-66). Similarly, Phase I clinicaltrials with ENZ-2208 revealed dose-limiting febrile neutropenia, with arecommendation to administer 10 mg/m² every 3 weeks or 16 mg/m² ifpatients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTCInternational Conference on Molecular Targets and Cancer Therapeutics;2009 Nov. 15-19; Boston, Mass.; Poster No C216; Patnaik et al.,AACR-NCI-EORTC International Conference on Molecular Targets and CancerTherapeutics; 2009 Nov. 15-19; Boston, Mass.; Poster No C221). Becausemonkeys tolerated a cumulative human equivalent dose of 22 mg/m², itappears that even though hRS7 binds to a number of normal tissues, theMTD for a single treatment of the hRS7 ADC could be similar to that ofthe other nontargeting SN-38 agents. Indeed, the specificity of theanti-Trop-2 antibody did not appear to play a role in defining the DLT,because the toxicity profile was similar to that of irinotecan. Moreimportantly, if antitumor activity can be achieved in humans as in micethat responded with human equivalent dose of just at 0.03 mg SN-38equivalents/kg/dose, then significant antitumor responses may berealized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 4. Anti-Trop-2 ADC Comprising hRS7 and Paclitaxel

A new antibody-drug conjugate (ADC) was made by conjugating paclitaxel(TAXOL®) to the hRS7 anti-human Trop-2 antibody (hRS7-paclitaxel). Thefinal product had a mean drug to antibody substitution ratio of 2.2.This ADC was tested in vitro using two different Trop-2-positive celllines as targets: BxPC-3 (human pancreatic adenocarcinoma) andMDA-MB-468 (human triple negative breast carcinoma). One day prior toadding the ADC, cells were harvested from tissue culture and plated into96-well plates at 2000 cells per well. The next day cells were exposedto free paclitaxel (6.1×10⁻¹¹ to 4×10⁻⁶ M) or the drug-equivalent ofhRS7-paclitaxel. For comparison, hRS7-SN-38 and free SN-38 were alsotested at a range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492nm) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

The hRS7-paclitaxel ADC exhibited cytotoxic activity in the MDA-MB-468breast cell line (FIG. 6), with an IC₅₀-value approximately 4.5-foldhigher than hRS7-SN-38. The free paclitaxel was much more potent thanthe free SN-38 (FIG. 6). While the IC₅₀ for free SN-38 was 1.54×10⁻⁹ M,the IC₅₀ for free paclitaxel was less than 6.1×10⁻¹¹ M. Similar resultswere obtained for the BxPC-3 pancreatic cell line (FIG. 7) in which thehRS7-paclitaxel ADC had an IC₅₀-value approximately 2.8-fold higher thanthe hRS7-SN-38 ADC. These results show the efficacy of anti-Trop-2conjugated paclitaxel in vitro, with IC₅₀-values in the nanomolar range,similar to the hRS7-SN-38 ADC.

Example 5. Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained for ADC conjugation. The first, 162-46.2, was purified from ahybridoma (ATCC, HB-187) grown up in roller-bottles. A second antibody,MAB650, was purchased from R&D Systems (Minneapolis, Minn.). For acomparison of binding, the Trop-2 positive human gastric carcinoma,NCI-N87, was used as the target. Cells (1.5×10⁵/well) were plated into96-well plates the day before the binding assay. The following morning,a dose/response curve was generated with 162-46.2, MAB650, and murineRS7 (0.03 to 66 nM). These primary antibodies were incubated with thecells for 1.5 h at 4° C. Wells were washed and an anti-mouse-HRPsecondary antibody was added to all the wells for 1 h at 4° C. Wells arewashed again followed by the addition of a luminescence substrate.Plates were read using Envision plate reader and values are reported asrelative luminescent units.

All three antibodies had similar K_(D)-values of 0.57 nM for RS7, 0.52nM for 162-46.2 and 0.49 nM for MAB650. However, when comparing themaximum binding (B_(max)) of 162-46.2 and MAB650 to RS7 they werereduced by 25% and 50%, respectively (B_(Max) 11,250 for RS7, 8,471 for162-46.2 and 6,018 for MAB650) indicating different binding propertiesin comparison to RS7.

Example 6. Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using twodifferent human pancreatic adenocarcinoma cell lines (BxPC-3 andCapan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto hRS7-SN-38, MAB650-SN-38, and free SN-38 at a drug range of3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a control atprotein equivalent doses as the MAB650-SN-38. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

As shown in FIG. 8, hRS7-SN-38 and MAB650-SN-38 had similargrowth-inhibitory effects with IC₅₀-values in the low nM range which istypical for SN-38-ADCs in these cell lines. In the human Capan-1pancreatic adenocarcinoma cell line (FIG. 8A), the hRS7-SN-38 ADC showedan IC₅₀ of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 1.0nM for free SN-38. In the human BxPC-3 pancreatic adenocarcinoma cellline (FIG. 8B), the hRS7-SN-38 ADC showed an IC₅₀ of 2.6 nM, compared to3.0 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38. In the humanNCI-N87 gastric adenocarcinoma cell line (FIG. 8C), the hRS7-SN-38 ADCshowed an IC₅₀ of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADCand 4.3 nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for hRS7-SN-38.

Example 7. Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to hRS7-SN-38, 162-46.2-SN-38, or free SN-38 at adrug range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated 162-46.2 and hRS7were used as controls at the same protein equivalent doses as the162-46.2-SN-38 and hRS7-SN-38, respectively. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492nm) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmnoidal dose response curves whichyield IC₅₀-values).

As shown in FIG. 9A and FIG. 9B, the 162-46.2-SN-38 ADC had a similarIC₅₀-values when compared to hRS7-SN-38. When tested against the BxPC-3human pancreatic adenocarcinoma cell line (FIG. 9A), hRS7-SN-38 had anIC₅₀ of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM forfree SN-38. When tested against the MDA-MB-468 human breastadenocarcinoma cell line (FIG. 9B), hRS7-SN-38 had an IC₅₀ of 3.9 nM,compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for free SN-38. Thefree antibodies alone showed little cytotoxicity to either Trop-2positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 8. Clinical Trials with IMMU-132 Anti-Trop-2 ADC Comprising hRS7Antibody Conjugated to SN-38

Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(˜1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients whohad failed multiple prior therapies (some including topoisomerase-I/IIinhibiting drugs), and the ongoing Phase II extension now reporting on69 patients, including in colorectal (CRC), small-cell and non-smallcell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC),pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≥2⁺) in most archived tumors. In a 3+3 trial design,IMMU-132 was given on days 1 and 8 in repeated 21-day cycles, startingat 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia.To optimize cumulative treatment with minimal delays, phase II isfocusing on 8 and 10 mg/kg (n=30 and 14, respectively). In 49 patientsreporting related AE at this time, neutropenia ≥G3 occurred in 28% (4%G4). Most common non-hematological toxicities initially in thesepatients have been fatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea(47%; ≥G3=9%), alopecia (40%), and vomiting (32%; ≥G3=2%). HomozygousUGT1A1 *28/*28 was found in 6 patients, 2 of whom had more severehematological and GI toxicities. In the Phase I and the expansionphases, there are now 48 patients (excluding PDC) who are assessable byRECIST/CT for best response. Seven (15%) of the patients had a partialresponse (PR), including patients with CRC (N=1), TNBC (N=2), SCLC(N=2), NSCLC (N=1), and esophageal cancers (N=1), and another 27patients (56%) had stable disease (SD), for a total of 38 patients (79%)with disease response; 8 of 13 CT-assessable PDC patients (62%) had SD,with a median time to progression (TTP) of 12.7 wks compared to 8.0weeks in their last prior therapy. The TTP for the remaining 48 patientsis 12.6+ wks (range 6.0 to 51.4 wks).

Plasma CEA and CA19-9 correlated with responses. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months. Theconjugate cleared from the serum within 3 days, consistent with in vivoanimal studies where 50% of the SN-38 was released daily, with >95% ofthe SN-38 in the serum being bound to the IgG in a non-glucoronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show that thehRS7-SN-38-containing ADC is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-3 8 in the serum was bound to the IgG. Lowconcentrations of SN-3 8G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient poll is provided in Table3.

TABLE 3 Clinical Trial Parameters Dosing Once weekly for 2 weeksadministered every 21 days regimen for up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥G2 treat-ment-related toxicity; protocol was amended to dose delay and reductiononly in the event of ≥G3 toxicity. Dose level 8, 12, 18 mg/kg; laterreduced to an intermediate cohorts dose level of 10 mg/kg. Cohort sizeStandard Phase I [3 + 3] design; expansion includes 15 patients inselect cancers. DLT G4 ANC ≥7 d; ≥G3 febrile neutropenia of anyduration; G4 Plt ≥5 d; G4 Hgb; Grade 4 N/V/D any duration/G3 N/V/Dfor >48 h; G3 infusion-related reactions; related ≥G3 non-hematologicaltoxicity. Maximum Maximum dose where ≥2/6 patients tolerate 1^(st) 21-dAcceptable cycle w/o delay or reduction or ≥G3 toxicity. Dose (MAD)Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast (TNBC), prostate, ovarian, renal,urinary bladder, head/neck, hepatocellular. Refractory/relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (FIG. 10) and Time to Progression (TTP; FIG.11) were determined. To summarize the Best Response data, of 8assessable patients with TNBC (triple-negative breast cancer), therewere 2 PR (partial response), 4 SD (stable disease) and 2 PD(progressive disease) for a total response [PR+SD] of 6/8 (75%). ForSCLC (small cell lung cancer), of 4 assessable patients there were 2 PR,0 SD and 2 PD for a total response of 2/4 (50%). For CRC (colorectalcancer), of 18 assessable patients there were 1 PR, 11 SD and 6 PD for atotal response of 12/18 (67%). For esophageal cancer, of 4 assessablepatients there were 1 PR, 2 SD and 1 PD for a total response of 3/4(75%). For NSCLC (non-small cell lung cancer), of 5 assessable patientsthere were 1 PR, 3 SD and 1 PD for a total response of 4/5 (80%). Overall patients treated, of 48 assessable patients there were 7 PR, 27 SDand 14 PD for a total response of 34/48 (71%). These results demonstratethat the anti-Trop-2 ADC (hRS7-SN-38) showed significant clinicalefficacy against a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 4. As apparent from the data of Table 4, the therapeutic efficacyof hRS7-SN-38 was achieved at dosages of ADC showing an acceptably lowlevel of adverse side effects.

TABLE 4 Related Adverse Events Listing for IMMU-132-01 Criteria: Total≥10% or ≥Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4(9%) 0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2(4%) Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0Dysgeusia 15% 0 0 Pyrexia 13% 0 0 Abdominal pain 11% 0 0 Hypokalemia 11%1 (2%) 0 WBC Decrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%)Deep vein thrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo 5FU. She presented with multiple lesionsprimarily in the liver (3+ Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of carboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topo-II inhibitor) that ended after 2 months with no response, followedwith topotecan (Topo-I inhibitor) that ended after 2 months, also withno response, she received local XRT (3000 cGy) that ended 1 month later.However, by the following month progression had continued. The patientstarted with IMMU-132 the next month (12 mg/kg; reduced to 6.8 mg/kg;Trop-2 expression 3+), and after two months of IMMU-132, a 38% reductionin target lesions, including a substantial reduction in the main lunglesion occurred (not shown). The patient progressed 3 months later afterreceiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies.

In conclusion, at the dosages used, the primary toxicity was amanageable neutropenia, with few Grade 3 toxicities. IMMU-132 showedevidence of activity (PR and durable SD) in relapsed/refractory patientswith triple-negative breast cancer, small cell lung cancer, non-smallcell lung cancer, colorectal cancer and esophageal cancer, includingpatients with a previous history of relapsing on topoisomerase-Iinhibitor therapy. These results show efficacy of the anti-Trop-2 ADC ina wide range of cancers that are resistant to existing therapies.

Example 9. Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM5 humanized MAb, hMN-14 (also known as labetuzumab), theanti-CD22 humanized MAb, hLL2 (also known as epratuzumab), the anti-CD20humanized MAb, hA20 (also known as veltuzumab), the anti-EGP-1 humanizedMAb, hRS7, and anti-mucin humanized MAb, hPAM4 (also known asclivatuzumab), were conjugated to SN-38 using a CL2A linker. Eachantibody was reduced with dithiothreitol (DTT), used in a 50-to-70-foldmolar excess, in 40 mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37° C.(bath) for 45 min. The reduced product was purified by size-exclusionchromatography and/or diafiltration, and was buffer-exchanged into asuitable buffer at pH 6.5. The thiol content was determined by Ellman'sassay, and was in the 6.5-to-8.5 SH/IgG range. Alternatively, theantibodies were reduced with Tris (2-carboxyethyl) phosphine (TCEP) inphosphate buffer at pH in the range of 5-7, followed by in situconjugation. The reduced MAb was reacted with ˜10-to-15-fold molarexcess of CL2A-SN-38 using DMSO at 7-15% v/v as co-solvent, andincubating for 20 min at ambient temperature. The conjugate was purifiedby centrifuged SEC, passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for these conjugates were typically in the 5-to-7 range.

Example 10. Combination Therapy with Anti-Trop-2 ADC in SCLC

Since cisplatinum is one of the main chemotherapeutics used incombination with irinotecan in advanced small-cell lung cancer (SCLC),an experiment was performed testing the combination of cisplatinum withIMMU-132 in mice bearing human SCLC tumors (DMS 53). Furthermore,carboplatin was tested in this tumor model since it too is usedclinically in SCLC.

Female C.B.-17 SCID mice were injected s.c. with a tumor suspension madefrom of DMS 53 stock tumors (20% w/v) plus cells harvested from tissueculture (5×10⁶ cells per mouse) mixed 1:1 with matrigel. Once tumorsreached a mean tumor volume of 0.270+0.048 cm³, the animals were dividedup into nine different treatment groups of 9 mice each. Three groups ofmice received IMMU-132 (500, 250 or 100 μg i.p.) while a control groupreceived a non-tumor targeting antibody-drug conjugate made with h679, ahumanized anti-histamine-succinyl-glycine IgG (h679-SN-38; 500 μg i.p.).All were administered twice weekly for 4 weeks. Two groups received onlychemotherapy of either cisplatinum (5 mg/kg i.p.) or carboplatin (50mg/kg i.p.) weekly for 4 weeks. Two groups received a combination ofIMMU-132 (250 μg i.v. weekly×4 wks) plus either cisplatinum orcarboplatin. A final untreated control group of mice received saline(100 μL i.p. twice weekly×4 wks). Tumors were measured and mice weighedtwice weekly.

Results

Mean tumor volumes for the various groups are shown in FIG. 12. Allthree doses of IMMU-132 provided a significant antitumor effect comparedto saline control animals (P<0.0161; AUC one-tailed t-test). Therapywith the highest dose of IMMU-132 (500 μg) produced significantlygreater tumor regressions in tumor-bearing mice compared to all themonotherapy groups, including control h679-SN-38 (P<0.0032; AUCtwo-tailed t-test).

For the combination groups, IMMU-132 (250 μg) plus carboplatin (FIG. 13)was approaching significance (P=0.0983; AUC two-tailed t-test) at thetime the first mouse in the control carboplatin monotherapy groupreached its end-point of tumor volume >1.0 cm³ on day 41 (therapy day14). However, in terms of absolute mean tumor volumes, on this day thecombination of IMMU-132 (250 μg) plus carboplatin had significantlysmaller tumors when compared to carboplatin monotherapy (0.166±0.019 cm³vs. 0.602±0.224 cm³, respectively; P=0.0004, two-tailed t-test). Whencompared to mice treated with only IMMU-132 (250 μg twice weekly), theIMMU-132 (weekly) plus carboplatin treated animals had significantlysmaller tumors as of day 73 (last day of comparison due to several micereaching end-point; 0.745+0.162 cm³ vs. 0.282±0.153 cm³, respectively;P=0.0003, AUC two-tailed t-test).

Mice treated with the combination of IMMU-132 (250 μg) plus cisplatinum(FIG. 14) exhibited significant tumor-growth inhibition when compared tocisplatinum monotherapy (P=0.0002, AUC two-tailed t-test). When tumorvolumes were compared on day 73 (last day of comparison due to severalmice in cisplatinum monotherapy group reaching end-point), thecombination group had tumors that were ˜6.2-fold smaller than those inthe cisplatinum monotherapy group (0.123±0.040 cm³ vs. 0.758±0.240 cm³,respectively; P<0.0001, two-tailed t-test). Likewise, mice treated withthe weekly schedule of IMMU-132 (250 μg) combined with cisplatinum hadsignificantly smaller tumors than animals treated with only IMMU-132(250 μg) even though in the monotherapy group IMMU-132 was administeredtwice weekly (P<0.0001, AUC two-tailed t-test). Finally, the combinationof IMMU-132 plus cisplatinum proved to be superior to all the othergroups including IMMU-132 plus carboplatin combination and high doseIMMU-132 (500 μg) monotherapy treatment groups (P<0.0066, AUC two-tailedt-test).

Both the IMMU-132 plus carboplatin treatment as well as the IMMU-132plus cisplatinum treatment were well tolerated by the mice. An unusualaspect of this tumor model was the observation that mice bearing DMS 53tumors exhibited cachexia (FIG. 15) with weights dropping on averagegreater than 15% from the start. For example, in the saline controlmice, as the tumor burden grew from 0.270±0.053 cm³ on day 27 to0.683±0.185 cm³ on day 41, the animal's body weight dropped 16.9%±3.4%.However, as the antitumor effects of IMMU-132 (500 μg) monotherapy orthe combinations of IMMU-132 plus carboplatin or cisplatinum becameevident, the mice began to gain weight again. For example, in theIMMU-132 plus carboplatin group, mice lost their greatest weight by day38 (15.3%±5.6%), after which, they began to regain this lost weight asthe tumors regressed with the mice returning to 100% of starting weightby day 58. During that time, the tumors regressed to 0.128±0.0.018 cm³from a starting size of 0.270±0.050 cm³ on day 27. However, animalsbegan to lose weight again as the tumors regrew to a size larger thanwhen therapy started (0.282+0.153 cm³ on day 73). In short, as thetumor-burden was decrease by these therapies, the debilitating effectsof cachexia were being reversed in these mice, further indicating thebenefit of these combinations in this human SCLC disease model.

Example 11. Therapy of Human mSCLC Patients with Anti-Trop-2-SN-38 ADC

Summary

Topotecan, a topoisomerase I inhibitor, is approved as a second-linetherapy in patients sensitive to first-line platinum-containingregimens, but no new therapeutic has been approved for the treatment ofmetastatic small-cell lung cancer (mSCLC) in twenty years. In thisExample, a novel antibody-drug conjugate (ADC), sacituzumab govitecan,comprised of an antibody targeting Trop-2 and containing the activemetabolite of irinotecan, SN-38 (also a topoisomerase I inhibitor), wasstudied. Patients with a median of 2 prior therapies (range 1-7)received the ADC on days 1 and 8 of 21-day cycles, with a median of tendoses (range, 1 to 63) being given. The principal grade ≥3 toxicitieswere manageable neutropenia, fatigue, and diarrhea. Despite up to 63repeated doses, the ADC was not immunogenic.

Forty-nine percent of the 43 assessable patients had a reduction oftumor size from baseline; the objective response rate (partialresponses) was 16% and stable disease was achieved in 49% of patients.Median progression-free survival and median overall survival were 3.6and 7.0 months, respectively, based on an intention-to-treat (N=53)analysis. This ADC was active in patients who were chemosensitive orchemoresistant to first-line chemotherapy, and also in patients whofailed second-line topotecan therapy. These data support the use ofsacituzumab govitecan as a new therapeutic for advanced mSCLC.

Methods

Patients ≥18 years of age with mSCLC who had relapsed or were refractoryto at least one prior standard line of therapy for stage IV metastaticdisease, and with measurable tumors by CT, were enrolled. They wererequired to have Eastern Cooperative Oncology Group (ECOG) performancestatus of 0 or 1, adequate bone marrow, hepatic and renal function, andother eligibility as described in the phase I trial (Starodub et al.,2015, Clin Cancer Res 21:3870-8). Previous therapy had to be completedat least 4 weeks before enrollment.

The overall objective of this portion of the basket trial beingconducted for diverse cancers (ClinicalTrials.gov, NCT01631552) was toevaluate safety and antitumor activity of sacituzumab govitecan inpatients with mSCLC. Sacituzumab govitecan was administeredintravenously at an initial infusion rate of 50 mg/h, completed within 3h (subsequent infusions completed within 60-90 min). Premedications(e.g., diphenhydramine, acetaminophen, and dexamethasone) wereprescribed optionally to reduce the risk of infusion reactions. Doses of8 or 10 mg/kg were given on days 1 and 8 of a 21-day cycle, withcontingencies to delay (maximum of 2 weeks). Toxicities were managed bysupportive hematopoietic growth-factor therapy for blood cell reduction,dose delays and/or modification as specified in the protocol (e.g., 25%of prior dose), or by standard medical practice. Treatment was continueduntil disease progression, initiation of alternative anticancer therapy,unacceptable toxicity, or withdrawal of consent.

Fifty-three patients were enrolled with mSCLC (30 females, 23 males,with a median age 63 years (range, 44-82). The median time from initialdiagnosis to treatment with sacituzumab govitecan was 9.5 months (range,3 to 53). Most patients were heavily pretreated, with a median of 2prior lines of therapy (range, 1 to 7). Everyone received cisplatinum orcarboplatin plus etoposide. Twenty-two (41%) patients had 1 prior lineof therapy, while 14 (26%) and 17 (32%) were given 2 and ≥3 priorchemotherapy regimens, respectively. In addition, 18 (33%) receivedtopotecan and/or irinotecan, 9 (16%) had a taxane, and 5 (9%) had animmune checkpoint inhibitor therapy, comprising nivolumab (N=4) oratezolizumab (N=1).

Based on a duration of response to a platinum-containing frontlinetherapy greater or less than 3 months, there were 27 (51%) and 26 (49%)chemosensitive and chemoresistant patients, respectively. Most patientshad extensive disease, with metastases to multiple organs, includinglungs (66%), liver (59%), lymph nodes (76%), chest (34%), adrenals(25%), bone (23%), and pleura (6%). Other sites of disease includedpancreas (N=4), brain (N=2), skin (N=2), and esophageal wall, ovary, andsinus (1 each).

The primary endpoint was the proportion of patients with a confirmedobjective response, assessed approximately every 8 weeks until diseaseprogression, by each institution's radiology group or a contracted localradiology service. Objective responses were assessed by ResponseEvaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1)(Eisenhauer et al., 2009, Eur J Cancer 45:228-47). Partial (PR) orcomplete responses (CR) required confirmation within 4 to 6 weeks afterthe initial response. Clinical benefit rate (CBR) is defined as thosepatients with an objective response plus stable disease (SD)≥4 months.Survival was monitored every 3 months until death or withdrawal ofconsent.

Safety evaluations were conducted during scheduled visits or morefrequently if warranted. Blood count and serum chemistries were checkedroutinely before administration of sacituzumab govitecan and whenclinically indicated.

Statistical Analyses—

The data included in the analyses were derived from patients enrolledfrom November 2013 to June 2016, with follow-up through Jan. 31, 2017.The frequency and severity of adverse events (AEs) were defined byMedDRA Preferred Term and System Organ Class (SOC) version 10, withseverity assessed by NCI-CTCAE v4.03. All patients who receivedsacituzumab govitecan were evaluated for toxicities.

The protocol provided that objective response rates (ORR) weredetermined for patients who received ≥2 doses (1 cycle) and had theirinitial 8-week CT assessment. Duration of response is defined inaccordance to RECIST 1.1 criteria, with those having an objectiveresponse marked from time of the first evidence of response untilprogression, while stable disease duration is marked from the start oftreatment until progression. PFS and OS are defined from the start oftreatment until an objective assessment of progression was determined(PFS) or death (OS). Duration of response, PFS, and OS were estimated byKaplan-Meier methods, with 95% confidence intervals (CI), using MedCalcStatistical Software, version 16.4.3 (Ostend, Belgium).

Tumor Trop-2 Immunohistochemistry and Immunogenicity of SacituzumabGovitecan and Components—

Archival tumor specimens for Trop-2 were stained by IHC and interpretedas reported previously (Starodub et al., 2015, Clin Cancer Res21:3870-8). Positivity required at least 10% of the tumor cells to bestained, with an intensity scored as 1+(weak), 2+(moderate), and3+(strong). Antibody responses to sacituzumab govitecan, the IgGantibody, and SN-38 were monitored in serum samples taken at baselineand then prior to each even-numbered cycle by enzyme-linkedimmunosorbent assays performed by the sponsor (Starodub et al., 2015,Clin Cancer Res 21:3870-8). Assay sensitivity is 50 ng/mL for the ADCand the IgG, and 170 ng/mL for anti-SN-38 antibody.

TABLE 5 Baseline demographics and disease characteristics of allpatients (N = 53) Age Years (median; range) 63 (44-82) Gender, N (%)Female 30 (56) Male 23 (44) Race, N (%) White 47 (88) Black 3 (6) Other3 (6) ECOG, N (%)  0 6 (11)  1 47 (89) Sites of metastases, Lung 35 (66)N (%) Liver 18 (59) Lymph nodes 40 (76) Chest 18 (34) Adrenals 13 (25)Bone 12 (23) Pleural effusion 3 (5) Pancreas 4 (7) Pelvis 2 (3) Brain 2(3) Skin 2 (3) Others 3 (5) Prior Lines of  1 22 (41) Therapy  2 14 (26)N (%) ≥3 17 (32) Sensitivity to 1^(st) line Sensitive 27 (51)chemotherapy, N (%) Resistant 26 (49) Prior Therapy, N (%) Platinum andEtoposide 53 (100) Topotecan and/or Irinotecan 18 (33) Taxanes 9 (16)Checkpoint inhibitors (CPI) 5 (9)

Results

Patients—

From November 2013 to June 2016, 53 patients were enrolled with mSCLC(30 females, 23 males, with a median age 63 years (range, 44-82) (Table5). The median time from initial diagnosis to treatment with sacituzumabgovitecan was 9.5 months (range, 3 to 53). Most patients were heavilypretreated, with a median of 2 prior lines of therapy (range, 1 to 7).Everyone received cisplatin or carboplatin plus etoposide. Twenty-two(41%) patients had 1 prior line of therapy, while 14 (26%) and 17 (32%)were given 2 and ≥3 prior chemotherapy regimens, respectively. Inaddition, 18 (33%) received topotecan and/or irinotecan, 9 (16%) had ataxane, and 5 (9%) had an immune checkpoint inhibitor therapy,comprising nivolumab (N=4) or atezolizumab (N=1). Based on a duration ofresponse to a platinum-containing frontline therapy greater or less than3 months, there were 27 (51%) and 26 (49%) chemosensitive andchemoresistant patients, respectively. Most patients had extensivedisease, with metastases to multiple organs, including lungs (66%),liver (59%), lymph nodes (76%), chest (34%), adrenals (25%), bone (23%),and pleura (6%) (Table 5). Other sites of disease included pancreas(N=4), brain (N=2), skin (N=2), and esophageal wall, ovary, and sinus (1each).

Treatment Exposure, Safety and Tolerability—

Of the 53 patients enrolled, two first treated in May 2016 werecontinuing sacituzumab govitecan therapy at the cutoff date of Jan. 31,2017. All other patients had discontinued treatment and otherwise werebeing monitored for survival. More than 590 doses (over 295 cycles) havebeen administered, with a median of 10 doses (range, 1-63) per patient.No infusion-related reactions were reported.

The initial doses in 15 patients were given at a starting dose of 8mg/kg; 10 mg/kg was the starting dose for the next 38 patients. Betweenthe 2 dose groups, 25 patients received ≥10 doses (≥5 cycles), and 2received 62 and 63 doses (>30 cycles). The median treatment duration was2.5 months (range, 1 to 23). Neutropenia (grade ≥2) was the onlyindication for dose reduction, and was recorded in 29% (11/38) patientsat the 10 mg/kg dose level after a median of 2.5 doses (range, 1 to 9).Two of the fifteen patients (13%) treated at 8 mg/kg had reductions, oneafter 2 doses and another after 41 doses (20 cycles). Once reduced,additional reductions were infrequent. No treatment-related deaths wereobserved.

In this trial, ten patients dropped out before the first responseassessment; four received 1 dose, five received 2 doses, and anotherafter 4 doses. Three were ineligible for response evaluation afterreceiving 1 or 2 doses, because one had mixed histology of SCLC andNSCLC, and the other 2 were diagnosed with pre-trial brain and/or spinalcord metastases after receiving the first dose of sacituzumab govitecan.Two patients who reported CTCAE grade 3 adverse events (neutropenia andfatigue) after one dose that did not recover in time for the second dosewere discontinued per protocol guidelines. Four patients withdrew fromthe study after 2 doses, 2 withdrew consent and 2 withdrew due to grade2 fatigue. An additional patient left the study after 4 treatmentsbecause of concurrent multiple comorbidities, dying suddenly before thefirst response assessment.

The most frequently reported AEs in the 53 patients receiving at leastone dose of sacituzumab govitecan were nausea, diarrhea, fatigue,alopecia, neutropenia, vomiting and anemia (Table 6). Grade 3 or 4neutropenia occurred in 34% (18/53) of patients, and only one patienthad febrile neutropenia. Other grade 3 or 4 adverse events were few, andincluded fatigue (13%), diarrhea (9%), anemia (8%), increased alkalinephosphatase (8%), and hyponatremia (8%). While there were fewer patientsrequiring dose reduction in the 8 mg/kg dose group (13% vs 28% in 10mg/kg), the 10 mg/kg dose level was equally well tolerated, with dosemodification and/or growth factor support in a few patients.

TABLE 6 Frequency of adverse events (N = 53), regardless of causality,occurring in >15% (all grades) or ≥2% (grade ≥3) of patients (ranked byall grades). Adverse Events All Grades Grades 3 and 4 (N = 53) N % N %Diarrhea 28 52.8 5 9.4 Nausea 27 50.9 — — Fatigue 25 47.2 7 13.2 Neutropenia 23 43.4 18  34.0  Vomiting 18 34.0 — — Abdominal pain 1630.2 — — Anorexia 15 28.3 — — Anemia 14 26.4 3 5.7 Alopecia 12 22.6 — —Constipation 11 20.8 — — Hypomagnesemia 10 18.9 — — Dehydration 9 17.0 —— Dyspnoea 9 17.0 — — Cough 8 15.1 — — Hypoxia 3 5.7 2 3.8 Febrileneutropenia 1 1.9 1 1.9

Efficacy—

As described, of the 53 mNSCLC patients enrolled, ten discontinued priorto their first CT response assessment, leaving 43 patients with theprotocol-required objective assessment of response after receiving atleast two doses of sacituzumab govitecan and at least one follow-upscan. FIG. 16 provides a series of graphic representations of theresponses, including a waterfall plot of the best percentage change inthe diameter sum of the target lesions for the 43 patients (FIG. 16A), agraph showing the duration of the responses for those achieving PR or SDstatus (FIG. 16B), and a plot tracking the response changes of thepatients with PR and SD over time (FIG. 16C).

Twenty-one of the 43 CT-assessable patients (49%) experienced areduction of tumor size from baseline (FIG. 16A). Confirmed partialresponses (≥30% reduction) occurred in seven patients, yielding an ORRof 16% (Table 7). The median time to response in these patients was 2.0months (range, 1.8 to 3.6 months), with a Kaplan-Meier estimated medianduration of response of 5.7 months (95% CI: 3.6, 19.9). Two of the sevenresponders had ongoing responses at the last follow-up (i.e., patientswere alive, free of disease progression, and had not started alternateanticancer treatments), one at 7.2+ months and the other 8.7+ monthsfrom start of treatment (FIG. 1B, FIG. 1C).

TABLE 7 Response summary of sacituzumab govitecan (IMMU-132) in SCLCpatients Best overall response, N (%) Total with response assessment 43PR (confirmed) 7 (16%) PRu (unconfirmed; SD with >30% shrinkage as 6(14%) best response) SD 15 (35%) PD 15 (35%) Clinical benefit rate (PR +SD ≥4 months) N (%) 17/43 (40%) Duration of confirmed objectiveresponse, 5.7 (3.6, 19.9) months median (95% CI) Progression-freesurvival, months (N = 53), 3.6 (2.0, 4.3) median (95% CI) Overallsurvival, months (N = 53), median 7.0 (5.5, 8.3) (95% CI) IMMU-132response assessment in patients who were sensitive (N = 24) to 1^(st)line. PFS (median months; 95% CI) OS (median months; 95% CI) 3.8 (2.8,6.0) Clinical benefit rate (PR + SD ≥4 months) N (%) 8.3 (7.0, 13.2)IMMU-1 32 response assessment in patients who were 12/24 (50%) resistant(N = 19) to 1^(st) line. PFS (median months; 95% CI) 3.6 (1.8, 3.8) OS(median months; 95% CI) 6.2 (4.0, 10.5) Clinical benefit rate (PR + SD≥4 months) N (%) 5/19 (26%) Patients receiving IMMU-132 as second line(N = 19) PFS, median months (95% CI) 3.6 (2.0, 5.3) OS (median months;95% CI) 8.1 (7.5, 10.5) Clinical benefit rate (PR + SD ≥4 months) N (%)7/19 (37%) Patients receiving IMMU-132 as ≥3 line (N = 24) PFS, medianmonths (95% CI) 3.7 (1.8, 5.5) OS (median months; 95% CI) 7.0 (6.2,20.9) Clinical benefit rate (PR + SD ≥4 months) N (%) 9/24 (38%)IMMU-132 given as ≥3 line and Prior topotecan/irinotecan (N = 15) PFS,median months (95% CI) 3.6 (3.3, 5.5) OS (median months; 95% CI) 8.8(6.2, 20.9) Clinical benefit rate (PR + SD ≥4 months) N (%) 6/15 (40%)No prior topotecan/irinotecan (N = 9) PFS, median months (95% CI) 3.7(1.7, 4.3) OS (median months; 95% CI) 5.5 (3.2, 8.3) Clinical benefitrate (PR + SD ≥4 months) N (%) 3/9 (33%)

Stable disease (SD) was determined in 21 patients (49%), and includedsix (14%) who initially had >30% tumor reduction that was not maintainedat the subsequent confirmatory CT (unconfirmed PR, or PRu), and threepatients who had ≥20% tumor reduction. It is important to note that tenpatients had SD for ≥4 months (Kaplan-Meier-derived median=5.6 months,95% CI: 5.2, 9.7), which was not significantly different from the medianPFS for the confirmed PR group (7.9 months, 95% CI: 7.6, 21.9;P=0.1620), and a clinical benefit rate (CBR: PR+SD≥4 months) of 40%(17/43). Indeed, even the OS for these ten SD patients was notsignificantly different from the seven confirmed PR patients (8.3months, 95% CI 7.5, 22.4 months vs 9.2 months, 95% CI: 6.2, 20.9,respectively; P=0.5599). This suggests that maintaining SD for asuitable duration (≥4 months) should be an endpoint of interest. On anintention-to-treat (ITT) basis (N=53), the median PFS was 3.6 months(95% CI: 2.0, 4.3) (FIG. 17 (A)), while the median OS was 7.0 months(95% CI: 5.5, 8.3), with 17 patients alive and 5 lost to follow-up (oneafter 1.8 months, one after 5 months, and three after 11.4-12.8 months)(FIG. 17 (B)).

Thirteen of the 43 patients with an objective response assessment weretreated at 8 mg/kg, with one confirmed (8%), one unconfirmed PR, andthree SD. In the 10 mg/kg group (N=30), six patients had confirmed PR(20%) and twelve had SD, including five with one CT showing a reduction≥30% (PRu). The CBR was 47% (14/30), suggesting that the starting doseof 10 mg/kg provided a better overall response.

Twenty-four patients with a response assessment were classified assensitive to the first line of platinum-based chemotherapy (Table 7).Four (17%) achieved a confirmed PR and nine had SD, including four witha single scan showing a >30% tumor reduction (PRu). Nineteen patientswere resistant, with three (16%) having confirmed PR and six with SD,including two with PRu. The median PFS for the chemosensitive andchemoresistant groups was 3.8 months (95% CI: 2.8, 6.0) and 3.6 months(95% CI: 1.8, 3.8), respectively, while the median OS was 8.3 months(95% CI: 7.0, 13.2) and 6.2 months (95% CI: 4.0, 10.5), respectively(Table 7). No significant differences in PFS or OS were found betweenthe chemosensitive and chemoresistant groups (P=0.3981 and P=0.3100,respectively).

Nineteen of the 43 patients received sacituzumab govitecan in thesecond-line setting, and 3/19 (16%) had a PR and seven SD as bestresponse (two of the latter had one >30% tumor shrinkage). The responseseen in these patients was the same as that found for the patients whowere given sacituzumab govitecan as their third or higher line oftherapy (N=24), with four confirmed PR (16%) and 8 SD, including four SDpatients with >30% tumor shrinkage on one CT. No significant differencesin duration of the PFS or OS were found (P=0.9538 and P=0.6853,respectively). Response analyses are summarized in Table 7.

Among the five patients who received prior treatment with an immunecheckpoint inhibitor (CPI), one experienced an unconfirmed PR (54%shrinkage on first assessment, withdrew consent without additionaltreatment or assessments), two achieved SD with one having 17% tumorshrinkage lasting 8.7 months and the other no change in tumor size for3.7 months, one had progressing disease, while the fifth patientwithdrew consent after one cycle of sacituzumab govitecan. All of theCPI-treated patients either failed to respond to the CPI or progressedbefore receiving sacituzumab govitecan, indicating that patients can beresponsive to IMMU-132 after receiving CPI-treatment.

Of the 24 patients who received sacituzumab govitecan as third- orlater-line therapy, fifteen had previously received topotecan and/oririnotecan, while nine never received these agents. The objectiveresponses in these two groups were similar, with no significantdifference in PFS (3.8 vs 3.7 months; P=0.7341). However, those treatedwith sacituzumab govitecan who received prior topotecan therapy had asignificantly longer OS than those who did not (8.8 months, 95% CI: 6.2,20.9 vs 5.5 months, 95% CI: 3.2, 8.3; P=0.0357). The longer OS in thisgroup may reflect the known activity of topotecan in patients who areplatinum-sensitive, and therefore may have a better long-term outcome.

A patient given sacituzumab govitecan after relapsing to first-linecarboplatin plus etoposide therapy is presented in FIG. 18. Anotherexample with considerable tumor reduction is shown in FIG. 19.

Immunohistochemical (IHC) Staining of Tumor Specimens—

Archival tumor specimens were obtained from 29 patients, but four wereinadequate for review, leaving 25 assessable tumors, of which 92% werepositive, with two (8%) having strong (3+) and thirteen (52%) moderate(2+) staining. Twenty-three of these patients had an objective responseassessment. There were five with confirmed PR and two unconfirmed PR inthis group; five had 2+ staining, while the other two were 1+(notshown), suggesting that higher expression provided better responses.However, an assessment of PFS and OS values against IHC score showed noclear trend (not shown), and Kaplan-Meier estimates for PFS and OS forpatients with IHC scores of 0 and 1+ combined (N=10) vs 2+ and 3+combined (N=13) indicated no significant differences (PFS, P=0.2661; OS,P=0.7186) based on IHC score (not shown).

Immunogenicity of ADC, SN-38, or hRS7 Antibody—

No neutralizing antibodies to sacituzumab govitecan, the antibody, orSN-38 were detected in patients who maintained treatment for even up to22 months.

Discussion

The relapse of SCLC to frontline chemotherapy continues to be dividedinto two categories, resistant relapse, occurring within three months ofthe first platinum-based therapy, and sensitive relapse, which occursafter at least 3 months post treatment (O'Brien et al., 2006, J ClinOncol 24:5441-7; Perez-Soler et al., 1996, J Clin Oncol 14:2785-90).Although there is still some ambiguity regarding the best management ofrecurrent SCLC, topotecan, a topoisomerase-I inhibitor similar to theSN-38 used in the ADC studied here, is the only product approved forchemosensitive relapse, as supported by numerous trials (O'Brien et al.,2006, J Clin Oncol 24:5441-7; Horita et al., 2015, Sci Rep 5:15437).However, the efficacy and adverse events of topotecan have variedconsiderably in prior studies, as demonstrated in a meta-analysis ofover a thousand patients reported in 14 articles that topotecan had anobjective response rate of 5% in chemoresistant frontline patients and17% in chemosensitive patients (Horita et al., 2015, Sci Rep 5:15437).There were grade ≥3 neutropenia, thrombocytopenia, and anemia in 69%,1%, and 24% of patients, respectively, and approximately 2% of patientsdied from this chemotherapy (Horita et al., 2015, Sci Rep 5:15437).Thus, topotecan shows some promise in this second-line setting inpatients who relapsed after showing sensitivity to a platinum-basedchemotherapy, but with considerable hematological toxicity. However,even this conclusion was challenged recently by Lara et al. (2015, JThorac Oncol 10:110-5), who asserted that platinum-sensitivity is notstrongly associated with improved PFS and OS following treatment withtopotecan, which is its currently approved indication.

It is in this setting that the results reported here with sacituzumabgovitecan in extended, advanced-disease patients (stage IV) following amedian of 2 (range, 1 to 7) prior therapies are promising. Forty-ninepercent of patients showed a reduction of tumor measurements frombaseline, according to RECIST 1.1, with an ORR of 16% and a medianduration of response of 5.7 months (95% CI: 3.6, 19.9). Stable diseasewas found in 35% of patients, where 14% of these SD patients had >30%tumor shrinkage as best response, although not maintained on the secondscan. The clinical benefit rate at ≥4 months was 40%. Median PFS and OSwere 3.6 and 7.0 months, respectively. It is interesting that the medianOS for the ten patients with SD was 8.3 months (95% CI: 7.5, 22.4),which is not statistically different from the median OS of 9.2 months(95% CI: 6.2, 20.9) for patients with a PR (P=0.5599). In the groupreceiving 10 mg/kg as their starting dose (N=30), there was a confirmedobjective response in six (20%), with an additional five patients havinga single CT showing ≥30% tumor reduction (PRu). Also, the clinicalbenefit rate for this group at the 10 mg/kg dose was 47%. This supportsthe preferred dose of 10 mg/kg. Noteworthy also is the lack of patientselection required based on immunohistochemical staining of tumorTrop-2, although there was a suggestion that stronger stainingcorrelated with better response, but no significant difference in PFS orOS was found with regard to IHC score.

As mentioned, PFS and OS did not differ substantially between patientswith SD>4 months or PR. Patients with unconfirmed PR (i.e., >30% tumorreduction on one CT) or with SD generally are not considered in most ORRassessments. However, the results here indicate no difference induration of response between patients with confirmed PR or SD lastingfor more than 4 months (FIG. 16B). Indeed, the dynamic tracking of theindividual patient responses for PR or SD (especially when the SD last≥4 months, which is a similar time frame for confirming PR) suggests aclinical benefit for both groups by remaining below the baseline tumorsize for several months (FIG. 16C). Although there was a trend for thePFS of patients with confirmed PR to be longer than the group ofpatients with SD lasting ≥4 months (P=0.1620), the OS for these 2 groupswas not significantly different (P=0.5599). Therefore, while the numberof patients in this initial analysis is relatively small, the datasuggest that more consideration should be given to disease stabilizationas an important indicator of clinical activity when an appropriateduration is achieved, similar to follow-up for patients receiving immunecheckpoint inhibitors.

Evaluating patients based on prior chemosensitivity (N=24) orchemoresistance (N=19) shows no response differences with sacituzumabgovitecan treatment (Table 7). PFS and OS results were 3.8 and 8.3months for patients who were chemosensitive in first-line, compared to aPFS and OS of 3.6 months and 6.2 months, respectively, for thechemoresistant group. With no statistical difference, it appears thatsacituzumab govitecan can be administered to patients in second- orlater-line therapies irrespective of the patients being chemosensitiveor chemoresistant to first-line chemotherapy. This differs fromtopotecan, which is indicated only in those SCLC patients who showed a≥3-month response to first-line cisplatin and etoposide chemotherapy((O'Brien et al., 2006, J Clin Oncol 24:5441-7; Perez-Soler et al.,1996, J Clin Oncol 14:2785-90). Of 28 patients studied by Perez-Solar etal. (1996, J Clin Oncol 14:2785-90), 11% had a PR, with a mediansurvival of 5 months and a one-year survival of 3.5%.

Although both topotecan and SN-38 are inhibitors of the DNAtopoisomerase I enzyme, which is responsible for relaxing a supercoiledDNA helix when DNA is synthesized by stabilizing the DNA complex,causing accumulation of single strand DNA breaks (Takimoto & Arbuck,1966, Camptothecins. In: Chabner & Long (Eds.). Cancer Chemotherapy andBiotherapy. Second ed. Philadelphia: Lippincott-Raven; p. 463-84),sacituzumab govitecan showed activity in patients who relapsed aftertopotecan therapy. Thus, topotecan resistance or relapse may not be acontraindication for administering sacituzumab govitecan, and because ofbeing similarly active in patients who were chemoresistant to cisplatinand etoposide, may be of particular value as a second-line therapeuticin patients with metastatic SCLC regardless of chemosensitivity status.

In the twenty years since the approval of topotecan in the second-linesetting, no new agent has been licensed for metastatic SCLC therapy insecond-line or later therapy. However, there has been progress morerecently with inhibitors of the T-cell checkpoint receptors programmedcell-death protein (PD-1) and cytotoxic T-lymphocyte-associated protein4 (CTLA-4) (Antonia et al., 2016, Lancet Oncol 17:883-95). These authorsconducted a phase I-II trial of nivolumab with or without CTLA-4antibody ipilimumab in patients with recurrent SCLC. Nivolumab aloneachieved a 10% response rate, while the combination had response ratesof 19 to 23%, and a disease-control rate of 32% (Antonia et al., 2016,Lancet Oncol 17:883-95). However, a recent study of ipilimumab with orwithout chemotherapy in SCLC failed to confirm these results (Reck etal., 2016, J Clin Oncol 34:3740-48). Since we observed that sacituzumabgovitecan may have activity in patients failing therapy with immunecheckpoint inhibitors, we are studying this further, especially becauseof evidence showing such responses after therapy with an immunecheckpoint inhibitor in patients with other cancer types (Bardia et al.,2017, J Clin Oncol 35:2141-48; Faltas et al., 2016, Clin GenitourinCancer 14:e75-9; Heist et al., 2017, J Clin Oncol 35:2790-97; Tagawa etal., 2017, J Clin Oncol 35:abstract 327).

Despite recent progress in immunotherapy and the identification of othernovel targets for SCLC (Rudin et al., 2017, Lancet Oncol 18:42-51), thisstill is a lethal disease, especially in the population that ischemoresistant to first-line therapy. The current results of sacituzumabgovitecan in heavily-pretreated patients with advanced, relapsed, stageIV, SCLC suggest that this ADC is of use in the therapy of bothchemosensitive and chemoresistant SCLC patients, both before or aftertopotecan.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of treating small cell lung cancer(SCLC) comprising administering to a human patient with SCLC ananti-Trop-2 antibody-drug conjugate (ADC) comprising SN-38 conjugated toan anti-Trop-2 antibody or antigen-binding fragment thereof.
 2. Themethod of claim 1, wherein the cancer is metastatic (mSCLC).
 3. Themethod of claim 1, wherein the ADC is administered as a first-linetherapy to patients who have not previously been treated for the SCLC.4. The method of claim 1, wherein the ADC is administered as asecond-line or later therapy to patients who have received previouscancer treatment for the SCLC.
 5. The method of claim 1, wherein thepatient has previously relapsed from or been resistant to treatment witha standard anti-cancer agent.
 6. The method of claim 1, wherein thepatient has previously relapsed from or been resistant to treatment withtopotecan or irinotecan.
 7. The method of claim 4, wherein the SCLC issensitive to chemotherapy with platinum-containing agents.
 8. The methodof claim 4, wherein the SCLC is resistant to chemotherapy withplatinum-containing agents.
 9. The method of claim 1, wherein the ADC isadministered at a dosage of between 4 mg/kg and 16 mg/kg.
 10. The methodof claim 9, wherein the dosage is selected from the group consisting of4 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, and 16mg/kg.
 11. The method of claim 9, wherein the dosage is between 8 mg/kgto 10 mg/kg.
 12. The method of claim 1, wherein the antibody is ahumanized RS7 antibody comprising the light chain CDR sequences CDR1(KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (NYGMN,SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO:6).
 13. The method of claim 1, wherein thetreatment results in a reduction in tumor size of at least 15%, at least20%, at least 30%, or at least 40%.
 14. The method of claim 2, furthercomprising reducing in size or eliminating the metastases.
 15. Themethod of claim 1, wherein the cancer is refractory to other therapiesbut responds to the ADC.
 16. The method of claim 1, wherein there is aCL2A linker between the SN-38 and the antibody and the structure of theADC is MAb-CL2A-SN-38


17. The method of claim 1, wherein there are 6 or more SN-38 moleculesattached to each antibody molecule.
 18. The method of claim 1, whereinthere are 6-8 SN-38 molecules attached to each antibody molecule. 19.The method of claim 1, wherein there are 7-8 SN-38 molecules attached toeach antibody molecule.
 20. The method of claim 1, wherein the antibodyis an IgG1 or IgG4 antibody.
 21. The method of claim 1, wherein theantibody has an allotype selected from the group consisting of G1m3,G1m3,1, G1m3,2, G1m3,1,2, nG1m1, nG1m1,2 and Km3 allotypes.
 22. Themethod of claim 1, wherein the ADC dosage is administered to the humansubject once or twice a week on a schedule with a cycle selected fromthe group consisting of: (i) weekly; (ii) every other week; (iii) oneweek of therapy followed by two, three or four weeks off; (iv) two weeksof therapy followed by one, two, three or four weeks off; (v) threeweeks of therapy followed by one, two, three, four or five weeks off;(vi) four weeks of therapy followed by one, two, three, four or fiveweeks off; (vii) five weeks of therapy followed by one, two, three, fouror five weeks off; and (viii) monthly.
 23. The method of claim 22,wherein the cycle is repeated 4, 6, 8, 10, 12, 16 or 20 times.
 24. Themethod of claim 1, wherein the ADC is administered in combination withone or more therapeutic modalities selected from the group consisting ofan unconjugated antibody, an immunoconjugate, gene therapy,chemotherapy, a therapeutic peptide, cytokine therapy, localizedradiation therapy, surgery, interference RNA therapy, a drug, a toxinand a cytokine.
 25. The method of claim 24, wherein the drug, toxin orchemotherapeutic agent is selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatinum (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel,dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine(2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin,erlotinib, entinostat, estrogen receptor binding agents, etoposide(VP16), etoposide glucuronide, etoposide phosphate, exemestane,fingolimod, flavopiridol, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR(FUdR-dO), fludarabine, flutamide, farnesyl-protein transferaseinhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide,imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 26. The method ofclaim 24, wherein the drug is cisplatinum or carboplatin.